{
  "21203919": {
    "bleu": 98.3206106870229,
    "meteor": 98.72717328332197,
    "rouge1_p": 0.9912739965095986,
    "rouge1_r": 0.9947460595446584,
    "rouge1_f": 0.9930069930069929,
    "rouge2_p": 0.9842657342657343,
    "rouge2_r": 0.987719298245614,
    "rouge2_f": 0.9859894921190893,
    "rougeL_p": 0.9912739965095986,
    "rougeL_r": 0.9947460595446584,
    "rougeL_f": 0.9930069930069929,
    "cosine_similarity": 0.9893129199292416,
    "precision": 0.9577464788732394,
    "recall": 0.9577464788732394,
    "ner_f1": 0.9577464788732394,
    "ner_tp": 68,
    "ner_fp": 3,
    "ner_fn": 3,
    "bertscore_p": 0.9907577633857727,
    "bertscore_r": 0.9907006025314331,
    "bertscore_f1": 0.9907664656639099,
    "bertscore_scibert_error": "The expanded size of the tensor (818) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 818].  Tensor sizes: [1, 512]",
    "len_gt": 3389,
    "len_jl": 3392,
    "GT": "MATERIALS AND METHODS Chemicals were purchased from Sigma-Aldrich, unless otherwise stated. DNA primers were also synthesized by Sigma-Aldrich (Supplemental Table 1). The P. aeruginosa PAO1 Washington strain was kindly provided by Dr Gail Preston, Department of Plant Sciences, University of Oxford, UK. paAzoR1, paAzoR2 and paAzoR3 were expressed and purified as described previously (Wang et al., 2007). Enzyme characterization was carried out as detailed previously (Wang et al., 2007). 2,6-dichlorindophenol (DCIP) reduction was measured via its absorbance at 595 nm and an extinction coefficient of 14,690 M\u207b\u00b9 cm\u207b\u00b9 was used. Reduction of menadione was measured, as described previously (Nakanishi et al., 2001), via monitoring the oxidation of NAD(P)H (absorbance at 340 nm). To measure accurately rates for menadione and azobenzene reduction 0.5 \u03bcg of enzyme was used per well, 150 \u03bcM NAD(P)H and 30\u20137.5 \u03bcM substrate in a total volume of 100 \u00b5L of 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl per well of a 96-well plate. Thermostability assays were carried out as described previously (Wang et al., 2010a), via measurement of enzyme activity versus methyl red after ten minutes incubation at defined temperatures. Quantitative RT-PCR Cultures of P. aeruginosa PAO1 were grown in LB medium containing 1 mM azo compound or 5-ASA to an absorbance at 600 nm of 1.0. A sample (5 mL) of each culture was removed RNA was extracted using an RNeasy Mini kit (Qiagen) followed by treatment with additional RNase-free DNase (Qiagen) to remove contaminating DNA. The concentration of RNA from each sample was adjusted to the same value for cDNA synthesis using a QuantiTect Reverse Transcription kit (Qiagen). Primers used in this study (RQ-785, RQ-962, RQ-223, rpoD, forward and reverse) were specifically designed to give PCR products around 150 base pair (Supplemental Table 1). Expression levels of paazor1, paazor2 and paazor3 were detected by quantitative RT-PCR analysis with a LightCycler (Roche) in comparison with expression of a P. aeruginosa \u03c3 factor rpoD (Savli et al., 2003). Each amplification mixture (10 \u03bcL QuantiTect 2 \u00d7 SYBR Green PCR master mix (Qiagen), 0.5 \u03bcg cDNA, 0.5 \u03bcM suitable primers and sterile water in a total 20 \u03bcL) was subjected to the following thermo-cycling program: one cycle of 95\u00b0C for 15 min to activate the HotStart Taq DNA polymerase; 40 cycles of denaturation (94\u00b0C for 15 s), annealing (60\u00b0C for 20 s), extension (72\u00b0C for 15 s) and data acquisition (65\u00b0C for 5 s). All reactions were performed in duplicate. In all PCR studies, RNA concentration was determined by measuring the absorbance at 260 nm and the integrity was confirmed by 1% agarose gel electrophoresis. Absence of DNA contamination was confirmed by running duplicate samples without reverse transcriptase as a negative control. Thin layer chromatography Thin layer chromatography (TLC) analysis (Sherma, 2003) was performed on a 0.1 mm thick silica gel-coated glass plate (Merck) using the mobile solvent system: tertiary amyl alcohol/formic acid/water (3:1:1, v/v) (Fetzner et al., 1992). Flavin co-factor was extracted from the protein component by denaturation (95\u00b0C, 10 min) and centrifugation (10,000 g, 5 min). Flavins were detected by direct visual observation of their yellow color. Authentic FMN, FAD and riboflavin were prepared in 20 mM Tris-HCl pH 8.0 buffer as standards.",
    "LLM": "MATERIALS AND METHODS Chemicals were purchased from Sigma-Aldrich, unless otherwise stated. DNA primers were also synthesized by Sigma-Aldrich (Supplemental Table 1). The P. aeruginosa PAO1 Washington strain was kindly provided by Dr Gail Preston, Department of Plant Sciences, University of Oxford, UK. paAzoR1, paAzoR2 and paAzoR3 were expressed and purified as described previously (Wang et al., 2007). Enzyme characterization was carried out as detailed previously (Wang et al., 2007). 2,6- dichlorindophenol (DCIP) reduction was measured via its absorbance at 595 nm and an extinction coefficient of 14,690 M-1$cm-1 was used. Reduction of menadione was measured, as described previously (Nakanishi et al., 2001), via monitoring the oxidation of NAD(P)H (absorbance at 340 nm). To measure accurately rates for menadione and azobenzene reduction 0.5 \u03bcg of enzyme was used per well, 150 \u03bcM NAD(P)H and 30\u20137.5 \u03bcM substrate in a total volume of 100 \u00b5L of 20mM Tris-HCl, pH 8.0, containing 100 mM NaCl per well of a 96- well plate. Thermostability assays were carried out as described previously (Wang et al., 2010a), via measurement of enzyme activity versus methyl red after ten minutes incubation at defined tempera\ufffetures. Quantitative RT-PCR Cultures of P. aeruginosa PAO1 were grown in LB medium containing 1 mM azo compound or 5-ASA to an absorbance at 600 nm of 1.0. A sample (5 mL) of each culture was removed RNA was extracted using an RNeasy Mini kit (Qiagen) followed by treatment with additional RNase-free DNase (Qiagen) to remove contaminating DNA. The concentration of RNA from each sample was adjusted to the same value for cDNA synthesis using a QuantiTect Reverse Transcription kit (Qiagen). Primers used in this study (RQ-785, RQ-962, RQ-223, rpoD, forward and reverse) were specifically designed to give PCR products around 150 base pair (Supplemental Table 1). Expression levels of paazor1, paazor2 and paazor3 were detected by quantitative RT-PCR analysis with a LightCycler (Roche) in comparison with expression of a P. aeruginosa \u03c3 factor rpoD (Savli et al., 2003). Each amplification mixture (10 \u03bcL QuantiTect 2 \u00d7 SYBR Green PCR master mix (Qiagen), 0.5 \u03bcg cDNA, 0.5 \u03bcM suitable primers and sterile water in a total 20 \u03bcL) was subjected to the following thermo-cycling program: one cycle of 95\u00b0C for 15 min to activate the HotStart Taq DNA polymerase; 40 cycles of denaturation (94\u00b0C for 15 s), annealing (60\u00b0C for 20 s), extension (72\u00b0C for 15 s) and data acquisition (65\u00b0C for 5 s). All reactions were performed in duplicate. In all PCR studies, RNA concentration was determined by measuring the absorbance at 260 nm and the integrity was confirmed by 1% agarose gel electrophoresis. Absence of DNA contamination was confirmed by running duplicate samples without reverse transcriptase as a negative control. Thin layer chromatography Thin layer chromatography (TLC) analysis (Sherma, 2003) was performed on a 0.1 mm thick silica gel-coated glass plate (Merck) using the mobile solvent system: tertiary amyl alcohol/formic acid/ water (3:1:1, v/v) (Fetzner et al., 1992). Flavin co-factor was extracted from the protein component by denaturation (95\u00b0C, 10 min) and centrifugation (10,000 g, 5 min). Flavins were detected by direct visual observation of their yellow color. Authentic FMN, FAD and riboflavin were prepared in 20 mM Tris-HCl pH 8.0 buffer as standards."
  },
  "24177163": {
    "bleu": 95.01283461679502,
    "meteor": 94.98674462506857,
    "rouge1_p": 0.9373977086743044,
    "rouge1_r": 0.9581764951902969,
    "rouge1_f": 0.9476732161323681,
    "rouge2_p": 0.8993041342611543,
    "rouge2_r": 0.9192468619246862,
    "rouge2_f": 0.909166149389613,
    "rougeL_p": 0.9373977086743044,
    "rougeL_r": 0.9581764951902969,
    "rougeL_f": 0.9476732161323681,
    "cosine_similarity": 0.8726412751202938,
    "precision": 0.8826086956521739,
    "recall": 0.9022222222222223,
    "ner_f1": 0.8923076923076922,
    "ner_tp": 203,
    "ner_fp": 27,
    "ner_fn": 22,
    "bertscore_p": 0.9429907202720642,
    "bertscore_r": 0.9581650495529175,
    "bertscore_f1": 0.9507471919059753,
    "bertscore_scibert_error": "The expanded size of the tensor (3483) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 3483].  Tensor sizes: [1, 512]",
    "len_gt": 14201,
    "len_jl": 14232,
    "GT": "2. Methods 2.1. Dyestuffs and chemicals Methyl red sodium salts, brilliant yellow and brilliant black BN were purchased from Sigma\u2013Aldrich (St. Louis, MO, USA). Methyl orange, brilliant crocein moo and 2-(40-hydroxyphenylazo) benzoic acid were supplied from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Ponceau S, ethyl red and other required chemicals were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). All chemicals used were analytical grade and of the highest purity available commercially. 2.2. Bacterial strains and plasmids The culture of B. laterosporus TISTR1911 was originally isolated from sediments collected near a wastewater effluent outlet of a local cotton textile factory (Kampaeng Saen Patana Co., Ltd., Nakhonpathom, Thailand). The pure culture was deposited in the Microbial Collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand and is maintained in glycerol (20% v/v) at 80 \u00b0C. Growth experiments were conducted aerobically at 30 \u00b0C and 150 rpm in a nutrient medium containing (per liter): 5 g of NaCl, 2 g of Bacto-peptone (BD Bioscience, MD, USA), and 1 g of yeast extract (BD Bioscience) for 48 h. The plasmid pET-28a(+) (Novagen, Madison, WI, USA) was used to clone the polymerase chain reaction (PCR) product corresponding to the brAzo gene (azoreductase gene). E. coli JM109 and E. coli BL21(DE3)RIL (Stratagene, La Jolla, CA, USA) were used for plasmid amplification and expression of the recombinant brAzo, respectively. E. coli was grown in Luria\u2013Bertani (LB) medium containing (per liter): 10 g of Bacto-tryptone (BD Bioscience), 5 g of yeast extract (BD Bioscience), and 5 g of NaCl supplemented with kanamycin (50 \u00b5g/mL) and chloramphenicol (30 \u00b5g/mL). 2.3. Identification of the microorganism Genomic DNA was isolated from B. laterosporus TISTR1911 by cell lysis using lysozyme (2 mg/mL), followed by freezing and thawing in 10 mM Tris\u2013HCl buffer containing 10 mM NaCl, pH 8.0. PCR was performed using the two universal primers for 16S rRNA gene amplification: forward, 5'-GAGTTTGATCCTGGCTCAG, and reverse, 3'-GGCTGGATCACCTCCTTTCT designed from base positions 186,357 to 187,764, respectively. The 16S rRNA gene sequence was analyzed using EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/ ; Kim et al., 2012) and a phylogenic tree was constructed using the neighbour-joining method (Saitou and Nei, 1987) with the Mega software (version 5.1; Tamura et al., 2011) based on a bootstrap analysis. The gene sequence was deposited in the DNA Data Bank of Japan (DDBJ) under the accession number AB817147. 2.4. Cloning of brAzo gene The nucleotide sequence of the predicted brAzo gene from B. laterosporus TISTR1911 was obtained based on the multiple alignments of conserved regions of genes encoding the FMN-dependent NADH-azoreductase-containing 236 amino acid residues of B. laterosporus LMG15441 (accession number EGP33505) and GI9 (CCF13845) and 5 strains of Bacillus and Brevibacillus sp. (accession numbers NP244911, YP003921798, YP083323, YP002769850, ZP20501409) in the National Center for Biotechnology Information (NCBI) database. The brAzo gene was amplified by PCR using genomic DNA as a template and the following primers: forward 5'-GATTACACATATGGCAAAAGTATTG-3' and reverse 5'-GTTTATTTTTCTCGAGCAAACGTTTTTGCG-3', designed to have restriction sites for NdeI and XhoI, respectively (underlined). The reverse primer was designed without a stop codon, which allowed the protein expression with a C-terminal His6 affinity tag. The PCR product was purified by agarose gel electrophoresis (1.5%, w/v) with the QIAEXII gel extraction kit from Qiagen (MD, USA) and digested with NdeI and XhoI (Takara Bio, Otsu, Japan) followed by ligation with a DNA Ligation Kit version 2 (Takara Bio) into the pET28a(+) vector to construct an expression vector with an His6 tag at its N- and C-termini. E. coli JM109 was transformed with the resulting plasmid. The brAzo-containing plasmid subsequently deleted its original Gly-codon adjacent to the gene encoding C-terminal His6 in the pET-28a vector in order to generate the correct codons for His6 translation. For this, site-directed mutagenesis was done using a PrimeSTAR Mutagenesis kit (Takara Bio) with the conditions following the manufacturer\u2019s instruction. The PCR mixture was used directly for the transformation of E. coli JM109 and then introduced into E. coli BL21(DE3)RIL by heat shock transformation. 2.5. Overexpression and purification of the recombinant BrAzo The recombinant E. coli BL21(DE3)RIL was cultured in 1 L of LB medium supplemented with antibiotics at 37 \u00b0C until the cell optical density at 600 nm reached \u22480.6. Heterologous gene expression was induced with 0.25 mM isopropyl \u03b2-D-thiogalactopyranoside (IPTG) after which the cells were further incubated at 25 \u00b0C for 20 h. Collected cells were disrupted by sonication using a Sonifier 250 (Branson, Danbury, CT, USA), and BrAzo was purified to homogeneity by nickel-chelating chromatography using Chelating Sepharose Fast Flow column (Amersham Biosciences, Uppsala, Sweden). Active fractions were combined, desalted using Sephadex G-25 (Amersham Biosciences) equilibrated with 25 mM HEPES buffer (pH 7.0) and dialyzed against the same buffer. All purification steps were performed at 4 \u00b0C. The protein content was determined by the Bradford method (Bradford, 1976) with bovine serum albumin as a standard. Protein concentration of purified BrAzo was determined by the amino acid content of the hydrolysate (6 N HCl for 24 h at 110 \u00b0C) using an Amino Tac JLC-500/V amino acid analyzer (JEOL, Tokyo, Japan), unless otherwise stated. The oxidative cleavage of double His6-tagged sequences was done using the modified procedure of Andberg et al. (2007). The fusion protein (114 \u00b5M) was chelated with 2 M equivalents of copper(II) sulfate for 2 min followed by the addition of ascorbate (65 mM) and hydrogen peroxide (5 mM). The reaction was incubated at 4 \u00b0C for 1 h in the dark. The reaction mixture was immediately loaded into the nickel-chelating Sepharose Fast Flow column to remove incomplete reaction products. The collected flow-through fraction was desalted by ultrafiltration using 10 MWCO (Vivaspin 20, Sartorius Stedim Biotech, Goettingen, Germany). The removal of His6 was confirmed using His-Detect In-Gel Stain reagent (Nacalai Tesque, Inc.) for visualizing the His6-tagged fusion protein directly in a polyacrylamide gel. 2.6. Azoreductase enzymatic assays The standard enzymatic assay (1 mL) was prepared in 67 mM of Britton\u2013Robinson (BR) buffer (0.1 M phosphoric acid, 0.1 M boric acid, and 0.1 M acetic acid titrated to the desired pH with 0.5 M NaOH) (at pH 6.0) containing 200 \u00b5M of nicotinamide adenine dinucleotide (reduced form; NADH), 50 \u00b5M of dye and pre-incubated at 30 \u00b0C for 5 min. The reaction was initiated by the addition of an appropriate amount of enzyme. The initial rates of dye decolorization were determined by monitoring the decrease in dye absorbance. One unit of azoreductase was defined as the amount of enzyme catalyzing the reduction of 1 \u00b5mol of dye per minute using a molar absorption coefficient (\u03b5430 nm) of 23,360 M\u207b\u00b9 cm\u207b\u00b9. Methyl orange was used to define the BrAzo units for the calibration of integrated NADH regeneration system in the packed-bed reactor designs in 2.8. For the determination of the substrate specificity for azo-dyes, the following extinction coefficients were used: \u03b5520 nm = 33,470 M\u207b\u00b9 cm\u207b\u00b9 for ponceau S, \u03b5465 nm = 23,250 M\u207b\u00b9 cm\u207b\u00b9 for methyl orange, \u03b5430 nm = 11,920 M\u207b\u00b9 cm\u207b\u00b9 for sudan orange G, \u03b5510 nm = 42,200 M\u207b\u00b9 cm\u207b\u00b9 for brilliant crocein moo, \u03b5570 nm = 56,500 M\u207b\u00b9 cm\u207b\u00b9 for brilliant black BN, \u03b5370 nm = 55,400 M\u207b\u00b9 cm\u207b\u00b9 for brilliant yellow, \u03b5450 nm = 13,400 M\u207b\u00b9 cm\u207b\u00b9 for ethyl red and \u03b5360 nm = 18,100 M\u207b\u00b9 cm\u207b\u00b9 for 2-(40-hydroxyphenylazo)benzoic acid. The last two dyes were prepared from a stock solution (5 mM) in ethanol, giving a final concentration of 1% ethanol in the assay reaction. The apparent maximal velocity (Vmax) and Michaelis\u2013Menten constant (Km) values were determined by a non-linear regression performed with Microsoft Excel version 2007 (Microsoft Corporation, Redlands, WA, USA). All values were determined at least in triplicate. 2.7. BrAzo biochemical characterization SDS\u2013PAGE of proteins was performed using Laemmli\u2019s method (Laemmli, 1970) and the 12% gel was stained with Rapid CBB KANTO (Kanto Kagaku, Tokyo, Japan). The molecular mass of the native form of double His6-tagged BrAzo was determined by a TSKgel G3000 SWXL size exclusion high performance liquid chromatography (HPLC) column (Tosoh, Tokyo, Japan, 7.8 \u00d7 300 mm). The elution was done with 50 mM HEPES buffer, pH 7.0, containing 150 mM NaCl at a flow rate of 0.7 mL/min and the detection was followed at 280 nm. The molecular mass standards used, included thyroglobulin (670 kDa), \u03b3-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa) (Bio-Rad Laboratories, Inc.). The molar ratio of the prosthetic group FMN bound to the BrAzo molecule was determined after denaturing the protein using SDS as described by Mendes et al. (2011b). The effect of pH on the BrAzo activity was determined over a range from pH 2.5 to pH 11.0 at 30 \u00b0C in 67 mM BR buffer using methyl red as substrate. The measurement of the optimum temperature of BrAzo was determined over a range of temperatures from 5 to 60 \u00b0C. All activity determinations were normalized to the highest activity recorded. Once, the optimal conditions were found, different cofactors and their combinations were tested (NADH, NADPH, FMN and FAD). The effect of NaCl (0\u2013500 mM) on the BrAzo activity was determined. For the determination of the BrAzo reaction mechanism, rates of reactions were measured by varying the concentrations of methyl red (from 5 to 25 \u00b5M) and NADH (50\u2013400 \u00b5M), while the concentration of the other substrate was kept constant. 2.8. Biodegradation of methyl orange in a packed-bed reactor A fixed-bed column with 1.0 cm diameter was packed using nickel-chelating Sepharose Fast Flow with a void volume (Vl) of 0.5 mL and a total bed volume (Vt) of 1 mL, followed by activation with 0.2 M nickel(II) sulfate solution (1 mL). After this, the resin was washed with distilled water (10 mL) and further equilibrated with 5 mL of cold 50 mM HEPES buffer (pH 7.0). Double His6-tagged BrAzo was immobilized on nickel-chelating Sepharose Fast Flow resin by loading 0.1 mg BrAzo (10 unitsmethyl red, equivalent to 0.2 unitsmethyl orange) solution through the bed followed by washing with 5 mL of cold 50 mM HEPES buffer (pH 7.0). The BrAzo bed column was equilibrated with 5 mL of 100 mM BR buffer (pH 6.0, 30 \u00b0C) before use. The substrate solutions (50 mL) containing 200 \u00b5M NADH and methyl orange (17\u2013170 \u00b5M) were kept at 30 \u00b0C and perfused through the sealed top of the column by circulation of temperature-controlled substrates using a peristaltic pump (Cassette tube pump SMP-21, Eyela, Tokyo, Japan) with a constant volumetric flow rate (Q) at 30, 60, 120, 240, 480, 600 mL/h. The eluted solution coming from the outlet was collected using a fraction collector (Bio-rad model 2128) and the absorbance was measured at 465 nm. Column performance was defined by the degree of dye conversion, P, the fraction of the substrate reacted in the reactor, calculated by (S0\u2013Si)/S0 where S0 and Si are the inlet and outlet substrate concentrations (\u00b5M), respectively. Space time (s) is the absolute residence time for dye flowing through the reactor, related to Vl and Q by: s = Vl/Q. The BrAzo stability in a batch reactor was determined by following the activity decrease of the free enzyme in a batch reactor incubated in 100 mM BR buffer, pH 6.0 at 30 \u00b0C. The deactivation rate constant (kd) was determined using an exponential decay model of E = E0exp(\u2212kdt), where E0 and E are initial and residual enzyme activities after different periods of time, respectively. The recycling PBR (packed-bed reactor) batch system was performed using the continuous flow reactor connected to the temperature controlled well-mixing reservoir (50 mL, 150 rpm stirring with a magnet) containing the substrate solution (S0 = 170 \u00b5M), which was fed continuously and circulated back from the reservoir into the PBR using the peristaltic pump. Sampling of a small portion of solution (50 \u00b5L) was done at desired time intervals to analyze the residual concentrations of substrate until the dye color disappeared. Two intermittent NADH-feeding profiles (0.5 and 1 h intervals) were compared using the constant Q = 120 mL/h. Reusability of the BrAzo bed was conducted by repeated use of the recycling PBR batch system at constant Q = 600 mL/h with 0.5 h-intermittent NADH-addition. At the end of each recycling batchwise cycle, the BrAzo bed was washed with 100 mM BR buffer at pH 6.0. For the next batchwise cycle, the reactor was filled again with a fresh substrate solution and the dye degradation process was then carried out several times with identical durations (3 h for each batch cycle). The set-up of an integrated system coupling free BrAzo with glucose 1-dehydrogenase (GDH; EC1.1.1.47) from Bacillus sp. (Wako Pure Chemical Industries Ltd.) was applied for the enzymatic reduction of methyl orange, where GDH regenerated the BrAzo substrate NADH. The basal reaction mixture (1 mL) contained 170 \u00b5M methyl orange, 67 mM BR buffer (pH 6.0) and BrAzo at 0.2 unitsmethyl red (equivalent to 0.004 unitsmethyl orange) at 30 \u00b0C using a polystyrene cuvette. Then, 5 min-intermittent NADH-additions (200 \u00b5M) were performed for the non-coupled reaction. The coupled reaction was performed by the addition of GDH at different ratios, 1:10, 1:15, and 1:20 (unitmethyl orange:unitGDH) in the basal solution supplemented with 200 \u00b5M \u03b2-nicotinamide adinine dinucleotide (NAD+) and 50 mM glucose. The reaction was followed by monitoring the absorbance at 465 nm for 1 h. The set-up of an integrated system with the two enzymes in a PBR recycling batchwise mode was prepared by a 50-fold scaling-up (50 mL reaction volume) using an identical recycling RBR batch system as described above. The dye removal process was assessed during 1.5 h for each batchwise cycle",
    "LLM": "2. Methods 2.1. Dyestuffs and chemicals Methyl red sodium salts, brilliant yellow and brilliant black BN were purchased from Sigma\u2013Aldrich (St. Louis, MO, USA). Methyl orange, brilliant crocein moo and 2-(40 -hydroxyphenylazo) benzoic acid were supplied from Wako Pure Chemical Industries Ltd. (To\ufffekyo, Japan). Ponceau S, ethyl red and other required chemicals were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). All chemi\ufffecals used were analytical grade and of the highest purity available commercially. 2.2. Bacterial strains and plasmids The culture of B. laterosporus TISTR1911 was originally isolated from sediments collected near a wastewater effluent outlet of a lo\ufffecal cotton textile factory (Kampaeng Saen Patana Co., Ltd., Nak\ufffehonpathom, Thailand). The pure culture was deposited in the Microbial Collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand and is maintained in glycerol (20% v/v) at 80 C. Growth experiments were conducted aerobically at 30 C and 150 rpm in a nutrient medium containing (per liter): 5 g of NaCl, 2 g of Bacto-peptone (BD Bioscience, MD, USA), and 1 g of yeast extract (BD Bioscience) for 48 h. The plasmid pET-28a(+) (Novagen, Madison, WI, USA) was used to clone the polymerase chain reaction (PCR) product corresponding to the brA\ufffezo gene (azoreductase gene). E. coli JM109 and E. coli BL21(DE3)RIL (Stratagene, La Jolla, CA, USA) were used for plasmid amplification and expression of the recombinant brAzo, respectively. E. coli was grown in Luria\u2013Bertani (LB) medium containing (per liter): 10 g of Bacto-tryptone (BD Bioscience), 5 g of yeast extract (BD Biosci\ufffeence), and 5 g of NaCl supplemented with kanamycin (50 lg/mL) and chloramphenicol (30 lg/mL). 2.3. Identification of the microorganism Genomic DNA was isolated from B. laterosporus TISTR1911 by cell lysis using lysozyme (2 mg/mL), followed by freezing and thawing in 10 mM Tris\u2013HCl buffer containing 10 mM NaCl, pH 8.0. PCR was performed using the two universal primers for 16S rRNA gene amplification: forward, 50 -GAGTTTGATCCTGGCTCAG, and reverse, 30 -GGCTGGATCACCTCCTTTCT designed from base positions 186,357 to 187,764, respectively. The 16S rRNA gene se\ufffequence was analyzed using EzTaxon-e server (http://eztaxon\ufffee.ezbiocloud.net/; Kim et al., 2012) and a phylogenic tree was con\ufffestructed using the neighbour-joining method (Saitou and Nei, 1987) with the Mega software (version 5.1; Tamura et al., 2011) based on a bootstrap analysis. The gene sequence was deposited in the DNA Data Bank of Japan (DDBJ) under the accession number AB817147. 2.4. Cloning of brAzo gene The nucleotide sequence of the predicted brAzo gene from B. lat\ufffeerosporus TISTR1911 was obtained based on the multiple align\ufffements of conserved regions of genes encoding the FMN\ufffedependent NADH-azoreductase-containing 236 amino acid resi\ufffedues of B. laterosporus LMG15441 (accession number EGP33505) and GI9 (CCF13845) and 5 strains of Bacillus and Brevibacillus sp. (accession numbers NP244911, YP003921798, YP083323, YP002769850, ZP20501409) in the National Center for Biotechnol\ufffeogy Information (NCBI) database. The brAzo gene was amplified by PCR using genomic DNA as a template and the following primers: forward 50 -GATTACACATATGGCAAAAGTATTG-30 and reverse 50 -GTTTATTTTTCTCGAGCAAACGTTTTTGCG-30 , designed to have restriction sites for NdeI and XhoI, respectively (underlined). The reverse primer was designed without a stop codon, which allowed the protein expression with a C-terminal His6 affinity tag. The PCR product was purified by agarose gel electrophoresis (1.5%, w/v) with the QIAEXII gel extraction kit from Qiagen (MD, USA) and di\ufffegested with NdeI and XhoI (Takara Bio, Otsu, Japan) followed by ligation with a DNA Ligation Kit version 2 (Takara Bio) into the pET28a(+) vector to construct an expression vector with an His6 tag at its N- and C-termini. E. coli JM109 was transformed with the resulting plasmid. The brAzo-containing plasmid subsequently deleted its original Gly-codon adjacent to the gene encoding C-ter\ufffeminal His6 in the pET-28a vector in order to generate the correct codons for His6 translation. For this, site-directed mutagenesis was done using a PrimeSTAR Mutagenesis kit (Takara Bio) with the conditions following the manufacturer\u2019s instruction. The PCR mixture was used directly for the transformation of E. coli JM109 and then introduced into E. coli BL21(DE3)RIL by heat shock transformation. 2.5. Overexpression and purification of the recombinant BrAzo The recombinant E. coli BL21(DE3)RIL was cultured in 1 L of LB medium supplemented with antibiotics at 37 C until the cell opti\ufffecal density at 600 nm reached \u00040.6. Heterologous gene expression was induced with 0.25 mM isopropyl b-D-thiogalactopyranoside (IPTG) after which the cells were further incubated at 25 C for 20 h. Collected cells were disrupted by sonication using a Sonifier 250 (Branson, Danbury, CT, USA), and BrAzo was purified to homo\ufffegeneity by nickel-chelating chromatography using Chelating Se\ufffepharose Fast Flow column (Amersham Biosciences, Uppsala, Sweden). Active fractions were combined, desalted using Sephadex G-25 (Amersham Biosciences) equilibrated with 25 mM HEPES buffer (pH 7.0) and dialyzed against the same buffer. All purifica\ufffetion steps were performed at 4 C. The protein content was deter\ufffemined by the Bradford method (Bradford, 1976) with bovine serum albumin as a standard. Protein concentration of purified BrAzo was determined by the amino acid content of the hydrolysate (6 N HCl for 24 h at 110 C) using an Amino Tac JLC-500/V amino acid ana\ufffelyzer (JEOL, Tokyo, Japan), unless otherwise stated. The oxidative cleavage of double His6-tagged sequences was done using the modified procedure of Andberg et al. (2007). The fu\ufffesion protein (114 lM) was chelated with 2 M equivalents of cop\ufffeper(II) sulfate for 2 min followed by the addition of ascorbate (65 mM) and hydrogen peroxide (5 mM). The reaction was incu\ufffebated at 4 C for 1 h in the dark. The reaction mixture was imme\ufffediately loaded into the nickel-chelating Sepharose Fast Flow column to remove incomplete reaction products. The collected flow-through fraction was desalted by ultrafiltration using 10 MWCO (Vivaspin 20, Sartorius Stedim Biotech, Goettingen, Ger\ufffemany). The removal of His6 was confirmed using His-Detect In\ufffeGel Stain reagent (Nacalai Tesque, Inc.) for visualizing the His6- tagged fusion protein directly in a polyacrylamide gel. 2.6. Azoreductase enzymatic assays The standard enzymatic assay (1 mL) was prepared in 67 mM of Britton\u2013Robinson (BR) buffer (0.1 M phosphoric acid, 0.1 M boric acid, and 0.1 M acetic acid titrated to the desired pH with 0.5 M NaOH) (at pH 6.0) containing 200 lM of nicotinamide adenine dinucleotide (reduced form; NADH), 50 lM of dye and pre-incu\ufffebated at 30 C for 5 min. The reaction was initiated by the addition of an appropriate amount of enzyme. The initial rates of dye decol\ufffeorization were determined by monitoring the decrease in dye absorbance. One unit of azoreductase was defined as the amount of enzyme catalyzing the reduction of 1 lmol of dye per minute using a molar absorption coefficient (e430 nm) of 23,360 M1 cm1 . Methyl orange was used to define the BrAzo units for the calibra\ufffetion of integrated NADH regeneration system in the packed-bed reactor designs in 2.8. For the determination of the substrate specificity for azo-dyes, the following extinction coefficients were used: e520 nm = 33,470 M1 cm1 for ponceau S, e465 nm = 23,250 M1 cm1 for methyl orange, e430 nm = 11,920 M1 cm1 for sudan orange G, e510 nm = 42,200 M1 cm1 for brilliant crocein moo, e570 nm = 56,500 M1 cm1 for brilliant black BN, e370 nm = 55,400 M1 cm1 for brilliant yellow, e450 nm = 13,400 M1 cm1 for ethyl red and e360 nm = 18,100 M1 cm1 for 2-(40 -hydroxyphenylazo)benzoic acid. The last two dyes were prepared from a stock solution (5 mM) in ethanol, giving a final concentration of 1% ethanol in the assay reaction. The apparent maximal velocity (Vmax) and Michaelis\u2013Menten constant (Km) values were determined by a non-linear regression performed with Microsoft\u0004 Excel version 2007 (Microsoft Corporation, Redlands, WA, USA). All values were determined at least in triplicate. 2.7. BrAzo biochemical characterization SDS\u2013PAGE of proteins was performed using Laemmli\u2019s method (Laemmli, 1970) and the 12% gel was stained with Rapid CBB KAN\ufffeTO (Kanto Kagaku, Tokyo, Japan). The molecular mass of the native form of double His6-tagged BrAzo was determined by a TSKgel G3000 SWXL size exclusion high performance liquid chromatogra\ufffephy (HPLC) column (Tosoh, Tokyo, Japan, 7.8 \u0005 300 mm). The elu\ufffetion was done with 50 mM HEPES buffer, pH 7.0, containing 150 mM NaCl at a flow rate of 0.7 mL/min and the detection was followed at 280 nm. The molecular mass standards used, included thyroglobulin (670 kDa), c-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa) (Bio\ufffeRad Laboratories, Inc.). The molar ratio of the prosthetic group FMN bound to the BrAzo molecule was determined after denatu\uffferating the protein using SDS as described by Mendes et al. (2011b). The effect of pH on the BrAzo activity was determined over a range from pH 2.5 to pH 11.0 at 30 C in 67 mM BR buffer using methyl red as substrate. The measurement of the optimum tem\ufffeperature of BrAzo was determined over a range of temperatures from 5 to 60 C. All activity determinations were normalized to the highest activity recorded. Once, the optimal conditions were found, different cofactors and their combinations were tested (NADH, NADPH, FMN and FAD). The effect of NaCl (0\u2013500 mM) on the BrAzo activity was determined. For the determination of the BrAzo reaction mechanism, rates of reactions were measured by varying the concentrations of methyl red (from 5 to 25 lM) and NADH (50\u2013400 lM), while the concentration of the other sub\ufffestrate was kept constant. 2.8. Biodegradation of methyl orange in a packed-bed reactor A fixed-bed column with 1.0 cm diameter was packed using nickel-chelating Sepharose Fast Flow with a void volume (Vl) of 0.5 mL and a total bed volume (Vt) of 1 mL, followed by activation with 0.2 M nickel(II) sulfate solution (1 mL). After this, the resin was washed with distilled water (10 mL) and further equilibrated with 5 mL of cold 50 mM HEPES buffer (pH 7.0). Double His6- tagged BrAzo was immobilized on nickel-chelating Sepharose Fast Flow resin by loading 0.1 mg BrAzo (10 unitsmethyl red, equivalent to 0.2 unitsmethyl orange) solution through the bed followed by washing with 5 mL of cold 50 mM HEPES buffer (pH 7.0). The BrAzo bed col\ufffeumn was equilibrated with 5 mL of 100 mM BR buffer (pH 6.0, 30 C) before use. The substrate solutions (50 mL) containing 200 lM NADH and methyl orange (17\u2013170 lM) were kept at 30 C and perfused through the sealed top of the column by circulation of temperature-controlled substrates using a peristaltic pump (Cassette tube pump SMP-21, Eyela, Tokyo, Japan) with a constant volumetric flow rate (Q) at 30, 60, 120, 240, 480, 600 mL/h. The eluted solution coming from the outlet was col\ufffelected using a fraction collector (Bio-rad model 2128) and the absorbance was measured at 465 nm. Column performance was defined by the degree of dye conversion, P, the fraction of the sub\ufffestrate reacted in the reactor, calculated by (S0Si)/S0 where S0 and Si are the inlet and outlet substrate concentrations (lM), respec\ufffetively. Space time (s) is the absolute residence time for dye flowing through the reactor, related to Vl and Q by: s = Vl/Q. The BrAzo stability in a batch reactor was determined by fol\ufffelowing the activity decrease of the free enzyme in a batch reactor incubated in 100 mM BR buffer, pH 6.0 at 30 C. The deactivation rate constant (kd) was determined using an exponential decay model of E = E0exp(kdt), where E0 and E are initial and residual enzyme activities after different periods of time, respectively. The recycling PBR (packed-bed reactor) batch system was performed using the continuous flow reactor connected to the temperature controlled well-mixing reservoir (50 mL, 150 rpm stirring with a magnet) containing the substrate solution (S0 = 170 lM), which was fed continuously and circulated back from the reservoir into the PBR using the peristaltic pump. Sam\ufffepling of a small portion of solution (50 lL) was done at desired time intervals to analyze the residual concentrations of substrate until the dye color disappeared. Two intermittent NADH-feeding profiles (0.5 and 1 h intervals) were compared using the constant Q = 120 mL/h. Reusability of the BrAzo bed was conducted by re\ufffepeated use of the recycling PBR batch system at constant Q = 600 mL/h with 0.5 h-intermittent NADH-addition. At the end of each recycling batchwise cycle, the BrAzo bed was washed with 100 mM BR buffer at pH 6.0. For the next batchwise cycle, the reac\ufffetor was filled again with a fresh substrate solution and the dye deg\uffferadation process was then carried out several times with identical durations (3 h for each batch cycle). The set-up of an integrated system coupling free BrAzo with glucose 1-dehydrogenase (GDH; EC1.1.1.47) from Bacillus sp. (Wako Pure Chemical Industries Ltd.) was applied for the enzy\ufffematic reduction of methyl orange, where GDH regenerated the BrAzo substrate NADH. The basal reaction mixture (1 mL) con\ufffetained 170 lM methyl orange, 67 mM BR buffer (pH 6.0) and BrAzo at 0.2 unitsmethyl red (equivalent to 0.004 unitsmethyl orange) at 30 C using a polystyrene cuvette. Then, 5 min-intermittent NADH-addi\ufffetions (200 lM) were performed for the non-coupled reaction. The coupled reaction was performed by the addition of GDH at differ\ufffeent ratios, 1:10, 1:15, and 1:20 (unitmethyl orange:unitGDH) in the ba\ufffesal solution supplemented with 200 lM b-nicotinamide adinine dinucleotide (NAD+ ) and 50 mM glucose. The reaction was fol\ufffelowed by monitoring the absorbance at 465 nm for 1 h. The set-up of an integrated system with the two enzymes in a PBR recycling batchwise mode was prepared by a 50-fold scal\ufffeing-up (50 mL reaction volume) using an identical recycling RBR batch system as described above. The dye removal process was as\ufffesessed during 1.5 h for each batchwise cycle."
  },
  "15870453": {
    "bleu": 90.24081115335868,
    "meteor": 93.39338469890029,
    "rouge1_p": 0.9538690476190477,
    "rouge1_r": 0.9591022443890275,
    "rouge1_f": 0.9564784879383238,
    "rouge2_p": 0.9196029776674938,
    "rouge2_r": 0.9246506986027944,
    "rouge2_f": 0.922119930330928,
    "rougeL_p": 0.9528769841269841,
    "rougeL_r": 0.9581047381546135,
    "rougeL_f": 0.9554837105197712,
    "cosine_similarity": 0.8936126597029732,
    "precision": 0.7653631284916201,
    "recall": 0.7210526315789474,
    "ner_f1": 0.7425474254742548,
    "ner_tp": 137,
    "ner_fp": 42,
    "ner_fn": 53,
    "bertscore_p": 0.9572242498397827,
    "bertscore_r": 0.9564830660820007,
    "bertscore_f1": 0.957027018070221,
    "bertscore_scibert_error": "The expanded size of the tensor (2837) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2837].  Tensor sizes: [1, 512]",
    "len_gt": 11465,
    "len_jl": 11487,
    "GT": "METHODS Bacterial strains, plasmids and growth conditions. S. aureus ATCC 25923 was grown in brain heart infusion (BHI) broth or on BHI agar plates and used for inoculum and genomic DNA preparation. E. coli TOP10F\u2032 (Invitrogen), NovaBlue (DE3) (Novagen), and BL21-Gold(DE3)pLysS (Stratagene) were used for recombinant DNA studies. E. coli strains were cultured at 37 \u00b0C in Luria\u2013Bertani (LB) medium with appropriate antibiotics (50 \u00b5g ml\u207b\u00b9). The plasmids pCR2.1-TOPO (Invitrogen) and pET-11d (Stratagene) were used for cloning and expression, respectively. Twenty millilitres of S. aureus from BHI broth was inoculated into 200 ml BHI medium supplemented with individual azo dye at a final concentration of 0.2 mM and incubated at 37 \u00b0C for various times in air without shaking. The supernatants were used to assay azo dye reduction by measuring residual absorption at the appropriate wavelength for each azo dye, as described in the enzyme assay description below. Cloning of S. aureus azo1 gene and expression of Azo1 in E. coli. Genomic DNA of S. aureus was isolated essentially as described by Wang et al. (2002). Plasmids from E. coli Top10F\u2032 and NovaBlue (DE3) were isolated using a Qiaprep Spin Miniprep kit (Qiagen). A DNA fragment containing the putative S. aureus azo1 was obtained by PCR with the genomic DNA of S. aureus used as template. The forward primer included a NcoI site before the start codon: 5\u2032-catgccatggctatgaaaggattaattattattggc-3\u2032 (Saa-forward). The reverse primer included a BamHI site downstream of the azo1 stop codon, beginning at 660 bp: 5\u2032-gcggatccgcttactattggtgcattag-3\u2032 (Saa-reverse). PCR was performed in a Mastercycler gradient (Eppendorf). The amplification conditions were one cycle at 95 \u00b0C for 3 min, 30 cycles with each cycle including 30 s of melting at 95 \u00b0C, 30 s of annealing at 50 \u00b0C and 60 s of extension at 72 \u00b0C, and one final extension cycle at 72 \u00b0C for 5 min. The PCR products were examined by 1 % agarose gel electrophoresis. The PCR products were directly cloned into the pCR2.1-TOPO vector and sequenced. Both strands of the DNA inserts were sequenced by walking from the ends of the inserts by using plasmid- and insert-specific primers. For overexpression of Azo1 in E. coli, the PCR products were cleaved with NcoI and BamHI (New England BioLabs). The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). E. coli NovaBlue (DE3) was transformed with the resulting plasmids. The plasmids (pAZO1) were subsequently isolated and introduced into E. coli BL21-Gold(DE3)pLysS by transformation. DNA sequence analysis, translation and alignment with related genes and proteins were carried out using the Lasergene program (Version 5, DNASTAR). The GenBank program BLAST was utilized to find similar genes or proteins. Enzyme assays. Azoreductase activity was assayed by measuring the decrease in optical density at suitable wavelengths with a Hewlett Packard 8453 UV\u2013visible spectrophotometer at 23 \u00b0C. The reaction mixture (2 ml) contained 25 mM potassium phosphate buffer (pH 7.1), 25 \u00b5M azo dye, 0.1 mM NADPH and a suitable amount of enzyme. The reaction was initiated by addition of the enzyme. Initial velocity was determined by monitoring the change in the amount of substrate in the first 2 min in a glass cuvette of 1 cm light path. The following molar absorption coefficients were used: 23,360 M\u207b\u00b9 cm\u207b\u00b9 (Methyl Red at 430 nm), 18,200 M\u207b\u00b9 cm\u207b\u00b9 (Orange II at 482 nm), 22,600 M\u207b\u00b9 cm\u207b\u00b9 (Amaranth at 520 nm), 20,700 M\u207b\u00b9 cm\u207b\u00b9 (Orange G at 477 nm), 22,900 M\u207b\u00b9 cm\u207b\u00b9 (Ponceau BS at 502 nm) (Nakanishi et al., 2001; Blumel et al., 2002) and 33,470 M\u207b\u00b9 cm\u207b\u00b9 (Ponceau S at 520 nm). One unit (U) of enzyme activity was defined as the amount of enzyme required to degrade one \u00b5mol azo dye per minute. Proteins were quantified using the bicinchoninic acid assay (Pierce) with BSA as standard. RNA isolation and RT-PCR. RNA was isolated from S. aureus grown overnight in BHI, BHI+Methyl Red (0.2 mM), or BHI+Orange II (0.2 mM), essentially using the RNAzol B procedure (Chapes et al., 1994). The RT-PCR reaction mixture (25 \u00b5l) consisted of 50 pmol each primer (Sas1-forward, 5\u2032-ggcagtgcacaagtgaattc-3\u2032 and Sas1-reverse, 5\u2032-ccatgatagtttggcgttcc-3\u2032), 4 \u00b5g RNA, 0.1 mM (each) deoxynucleoside triphosphate, 1 U Taq DNA polymerase and 10 U AMV reverse transcriptase in PCR buffer with MgCl\u2082. cDNA synthesis by reverse transcription was accomplished at 37 \u00b0C for 1 h. The PCR was performed under the following conditions: initial denaturation (95 \u00b0C, 2 min), 40 cycles of denaturation (94 \u00b0C, 1 min), annealing (55 \u00b0C, 1.5 min) and extension (72 \u00b0C, 1 min), followed by a 10 min final extension (72 \u00b0C). The reaction products were analysed by 2 % agarose gel electrophoresis. Enzyme purification. Induction of target protein and preparation of cell extracts were performed using a similar procedure to that described previously (Chen et al., 2004). Protein purification was performed at 4 \u00b0C by using an AKTApurifier 10 system with UNICORN 4.10 software (Amersham Biosciences). Sixty millilitres of the supernatant (549 mg protein) was applied to a HiPrep 16/10 Q XL anion-exchange column (Amersham Biosciences) equilibrated with 25 mM Tris/HCl (pH 7.5) buffer. The peak fractions of azoreductase activity eluted at around 0.3 M NaCl (21 ml) were pooled. One volume of 10 mM potassium phosphate (pH 7.1) containing 2.0 M (NH\u2084)\u2082SO\u2084 was mixed with one volume of the pooled fractions. Forty millilitres of the sample was applied to a HiPrep 16/10 Phenyl FF hydrophobic interaction column (Amersham Biosciences) equilibrated with the same buffer plus 1.0 M (NH\u2084)\u2082SO\u2084. A linear gradient of 400 ml (NH\u2084)\u2082SO\u2084 from 1.0 to 0 M in the same buffer was applied, followed by further elution with 100 ml buffer. Fractions with azoreductase activity (30 ml) were pooled and concentrated to a volume of about 8 ml by using an Amicon Ultra-15 (Millipore). The sample was dialysed and buffered with 25 mM Tris/HCl (pH 8.0). The sample was then applied in 2 ml portions to a Mono Q HR 5/5 column (Amersham Biosciences) equilibrated with the same buffer. Fractions of 0.5 ml were collected and those containing major enzyme activity were pooled (1.0 ml, 0.35 M NaCl). Final purification was achieved by gel filtration on a HiLoad 26/60 Superdex 200 pre grade column (Amersham Biosciences) equilibrated and eluted with 25 mM potassium phosphate (pH 7.1) plus 0.1 M NaCl (Chen et al., 2004). Identification of the prosthetic group. One hundred microlitres of the purified enzyme (3 mg ml\u207b\u00b9) in 10 mM potassium phosphate buffer (pH 7.0) was heated at 100 \u00b0C for 10 min in the dark. After cooling on ice, the denatured protein was removed by centrifugation at 14,500 g for 10 min in a microfuge at 4 \u00b0C. The resulting yellow supernatant (5 \u00b5l) was analysed by TLC employing a silica gel 60 F-254 plate (2 mm thickness; Merck) and chloroform, glacial acetic acid and water (6 : 7 : 1 by vol.) as the solvent phase (Lake & Goodwin, 1976). FAD and FMN (1 \u00b5l, 0.2 mM each) were used as standards, and the migration of the compounds was monitored by the characteristic fluorescence in UV light (312 nm). SDS-PAGE and N-terminal amino acid sequencing of the protein. SDS-PAGE (12.5 %, w/v) was conducted under normal conditions. Protein markers (Novagen) were used as standards. Gels were stained for the presence of proteins using Coomassie brilliant blue R-250 (Bio-Rad). The proteins were transferred onto a PVDF membrane in a Hoefer TE77 Semi-Dry Transfer Unit (Amersham Biosciences). The transferred proteins were visualized by Coomassie staining and excised with a razor blade. N-terminal amino acid sequencing was performed by an Applied Biosystems model 477A gas-phase sequencer equipped with an automatic on-line phenylthiohydantoin analyser. Preparation of apoenzyme and reconstruction of flavoprotein. Eight millilitres of 25 mM potassium phosphate (pH 7.1) containing 1 M (NH\u2084)\u2082SO\u2084, 1 M KBr and 1 mM EDTA was mixed with 2 ml purified enzyme (5 mg). The sample was applied to the HiPrep 16/10 Phenyl FF column equilibrated with the same buffer. The column was washed with 40 ml starting buffer. Flavin was eluted with 60 ml of the same buffer adjusted to pH 4.0 with 1 M phosphoric acid. After flavin elution, the column was immediately washed with 40 ml starting buffer, omitting 1 M KBr. Four hundred millilitres of a linear gradient of (NH\u2084)\u2082SO\u2084 from 1 to 0 M in the same buffer was applied. This was followed by further elution with 100 ml starting buffer. The fractions (30 ml) containing apoenzyme were concentrated (Van Berkel et al., 1988). The protein was reconstructed directly on the column. After the release of the original flavin from the column, the pH of the column was equilibrated back to 7.1, as described above. Forty millilitres of the starting buffer containing 1 mM FMN, FAD, or riboflavin was passed through the column at a flow rate of 0.5 ml min\u207b\u00b9. The column was placed at 4 \u00b0C overnight and then washed with 40 ml starting buffer, omitting 1 M KBr, to remove the unbound excess flavin. The elution of the protein was done as above (Van Berkel et al., 1988). Identification of Methyl Red and its metabolites. Two assay mixtures containing 0.4 mM NADPH and 50 \u00b5M Methyl Red in 20 ml of 25 mM sodium phosphate buffer (pH 7.1) with or without the purified enzyme were prepared. Methyl Red, N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid (Sigma) solutions (50 mg l\u207b\u00b9), using acetonitrile as solvent, were prepared. The assay mixture containing the enzyme was then incubated at room temperature until no decrease in optical density at 430 nm was noted. The two assay mixtures were extracted three times with equal volumes of ethyl acetate, subsequent to adjusting the pH to 3 with 1 M HCl. The extracts were evaporated in a rotary evaporator at 40 \u00b0C and the remaining trace solvent was removed by evaporation at room temperature overnight. Each residue was dissolved in 1 ml acetonitrile and filtered through a 0.2 \u00b5m pore-size syringe filter. Forty microlitres of the samples was analysed with a Hewlett Packard HPLC 1050 equipped with a model variable-wavelength detector (detection wavelength was 250 nm) and a reverse-phase Inertsil 5 \u00b5m ODS-2 column (4.6 \u00d7 250 mm, MetaChem Technologies). The mobile phase was composed of 25 mM phosphate buffer (pH 3.0) and acetonitrile (4 : 6, v/v) with a flow rate of 0.5 ml min\u207b\u00b9 (Moutaouakkil et al., 2003; Nakanishi et al., 2001; Wong & Yuen, 1998). Enzyme characterization. Each data point was analysed three times with a standard deviation of less than 10 %. The effects of pH on the activity and the optimum pH of S. aureus Sa-Azo1 were determined at room temperature with the following buffers: 0.1 M sodium acetate (pH 4.2\u20135.4), sodium phosphate (pH 5.8\u20137.8) and HEPES/NaOH (pH 8.2 and 8.6). The effects of temperature on Azo1 were determined by assaying the enzyme at temperatures ranging from 10 to 65 \u00b0C. Thermostability was measured by incubating the enzyme samples in 25 mM potassium phosphate buffer, pH 7.1, for 1 h at temperatures from 30 to 70 \u00b0C. The initial velocities of the enzymic reaction were obtained by varying the concentrations of one substrate, Methyl Red (from 0.005 to 0.04 mM) or NADPH (from 0.05 to 0.4 mM), while the concentration of the other substrate was kept constant (NADPH, 0.4 mM or Methyl Red, 0.04 mM). Apparent Km and Vmax values were obtained from Lineweaver\u2013Burk plots",
    "LLM": "METHODS Bacterial strains, plasmids and growth conditions. S. aureus ATCC 25923 was grown in brain heart infusion (BHI) broth or on BHI agar plates and used for inoculum and genomic DNA prepara\ufffetion. E. coli TOP10F9 (Invitrogen), NovaBlue (DE3) (Novagen), and BL21-Gold(DE3)pLysS (Stratagene) were used for recombinant DNA studies. E. coli strains were cultured at 37 uC in Luria\u2013Bertani (LB) medium with appropriate antibiotics (50 mg ml21 ). The plasmids pCR2.1-TOPO (Invitrogen) and pET-11d (Stratagene) were used for cloning and expression, respectively. Twenty millilitres of S. aureus from BHI broth was inoculated into 200 ml BHI medium supplemented with individual azo dye at a final concentration of 0?2 mM and incubated at 37 uC for various times in air without shaking. The supernatants were used to assay azo dye reduction by measuring residual absorption at the appropriate wavelength for each azo dye, as described in the enzyme assay description below. Cloning of S. aureus azo1 gene and expression of Azo1 in E. coli. Genomic DNA of S. aureus was isolated essentially as described by Wang et al. (2002). Plasmids from E. coli Top10F9 and NovaBlue (DE3) were isolated using a Qiaprep Spin Miniprep kit (Qiagen). A DNA fragment containing the putative S. aureus azo1 was obtained by PCR with the genomic DNA of S. aureus used as template. The forward primer included a NcoI site before the start codon: 59-catgccatggctatgaaaggattaattattattggc-39 (Saa-forward). The reverse primer included a BamHI site downstream of the azo1 stop codon, beginning at 660 bp: 59-gcggatccgcttactattggtgcattag-39 (Saa-reverse). PCR was performed in a Mastercycler gradient (Eppendorf). The amplification conditions were one cycle at 95 uC for 3 min, 30 cycles with each cycle including 30 s of melting at 95 uC, 30 s of annealing at 50 uC and 60 s of extension at 72 uC, and one final extension cycle at 72 uC for 5 min. The PCR products were examined by 1 % agarose gel electrophoresis. The PCR products were directly cloned into the pCR2.1-TOPO vector and sequenced. Both strands of the DNA inserts were sequenced by walking from the ends of the inserts by using plasmid- and insert\ufffespecific primers. For overexpression of Azo1 in E. coli, the PCR products were cleaved with NcoI and BamHI (New England BioLabs). The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). E. coli NovaBlue (DE3) was transformed with the resulting plasmids. The plasmids (pAZO1) were subsequently isolated and introduced into E. coli BL21-Gold(DE3)pLysS by transformation. DNA sequence analysis, translation and alignment with related genes and proteins were carried out using the Lasergene program (Version 5, DNASTAR). The GenBank program BLAST was utilized to find similar genes or proteins. Enzyme assays. Azoreductase activity was assayed by measuring the decrease in optical density at suitable wavelengths with a Hewlett Packard 8453 UV\u2013visible spectrophotometer at 23 uC. The reaction mixture (2 ml) contained 25 mM potassium phosphate buffer (pH 7?1), 25 mM azo dye, 0?1 mM NADPH and a suitable amount of enzyme. The reaction was initiated by addition of the enzyme. Initial velocity was determined by monitoring the change in the amount of substrate in the first 2 min in a glass cuvette of 1 cm light path. The following molar absorption coefficients were used: 23 360 M21 cm21 (Methyl Red at 430 nm), 18 200 M21 cm21 (Orange II at 482 nm), 22 600 M21 cm21 (Amaranth at 520 nm), 20 700 M21 cm21 (Orange G at 477 nm), 22 900 M21 cm21 (Ponceau BS at 502 nm) (Nakanishi et al., 2001; Blumel et al., 2002) and 33 470 M21 cm21 (Ponceau S at 520 nm). One unit (U) of enzyme activity was defined as the amount of enzyme required to degrade one mmol azo dye per minute. Proteins were quantified using the bicinchoninic acid assay (Pierce) with BSA as standard. RNA isolation and RT-PCR. RNA was isolated from S. aureus grown overnight in BHI, BHI+Methyl Red (0?2 mM), or BHI+ Orange II (0?2 mM), essentially using the RNAzol B procedure (Chapes et al., 1994). The RT-PCR reaction mixture (25 ml) con\ufffesisted of 50 pmol each primer (Sas1-forward, 59-ggcagtgcacaagt\ufffegaattc-39 and Sas1-reverse, 59-ccatgatagtttggcgttcc-39), 4 mg RNA, 0?1 mM (each) deoxynucleoside triphosphate, 1 U Taq DNA poly\ufffemerase and 10 U AMV reverse transcriptase in PCR buffer with MgCl2. cDNA synthesis by reverse transcription was accomplished at 37 uC for 1 h. The PCR was performed under the following condi\ufffetions: initial denaturation (95 uC, 2 min), 40 cycles of denaturation (94 uC, 1 min), annealing (55 uC, 1?5 min) and extension (72 uC, 1 min), followed by a 10 min final extension (72 uC). The reaction products were analysed by 2 % agarose gel electrophoresis. Enzyme purification. Induction of target protein and preparation of cell extracts were performed using a similar procedure to that described previously (Chen et al., 2004). Protein purification was performed at 4 uC by using an AKTApurifier 10 system with UNICORN 4.10 software (Amersham Biosciences). Sixty millilitres of the supernatant (549 mg protein) was applied to a HiPrep 16/10 Q XL anion-exchange column (Amersham Biosciences) equilibrated with 25 mM Tris/HCl (pH 7?5) buffer. The peak fractions of azoreductase activity eluted at around 0?3 M NaCl (21 ml) were pooled. One volume of 10 mM potassium phosphate (pH 7?1) containing 2?0 M (NH4)2SO4 was mixed with one volume of the pooled fractions. Forty millilitres of the sample was applied to a HiPrep 16/10 Phenyl FF hydrophobic interaction column (Amersham Biosciences) equilibrated with the same buffer plus 1?0 M (NH4)2SO4. A linear gradient of 400 ml (NH4)2SO4 sulfate from 1?0 to 0 M in the same buffer was applied, followed by further elution with 100 ml buffer. Fractions with azoreductase activity (30 ml) were pooled and concentrated to a volume of about 8 ml by using an Amicon Ultra-15 (Millipore). The sample was dialysed and buffered with 25 mM Tris/HCl (pH 8?0). The sample was then applied in 2 ml portions to a Mono Q HR 5/5 column (Amersham Biosciences) equilibrated with the same buffer. Fractions of 0?5 ml were collected and those containing major enzyme activity were pooled (1?0 ml, 0?35 M NaCl). Final purification was achieved by gel filtration on a HiLoad 26/60 Superdex 200 pre grade column (Amersham Biosciences) equilibrated and eluted with 25 mM potas\ufffesium phosphate (pH 7?1) plus 0?1 M NaCl (Chen et al., 2004). Identification of the prosthetic group. One hundred microlitres of the purified enzyme (3 mg ml21 ) in 10 mM potassium phosphate buffer (pH 7?0) was heated at 100 uC for 10 min in the dark. After cooling on ice, the denatured protein was removed by centrifugation at 14 500 g for 10 min in a microfuge at 4 uC. The resulting yellow supernatant (5 ml) was analysed by TLC employing a silica gel 60 F-254 plate (2 mm thickness; Merck) and chloroform, glacial acetic acid and water (6 : 7 : 1 by vol.) as the solvent phase (Lake & Goodwin, 1976). FAD and FMN (1 ml, 0?2 mM each) were used as standards, and the migration of the compounds was monitored by the characteristic fluorescence in UV light (312 nm). SDS-PAGE and N-terminal amino acid sequencing of the protein. SDS-PAGE (12?5 %, w/v) was conducted under normal conditions. Protein markers (Novagen) were used as standards. Gels were stained for the presence of proteins using Coomassie brilliant blue R-250 (Bio-Rad). The proteins were transferred onto a PVDF membrane in a Hoefer TE77 Semi-Dry Transfer Unit (Amersham Biosciences). The transferred proteins were visualized by Coomassie staining and excised with a razor blade. N-terminal amino acid sequencing was performed by an Applied Biosystems model 477A gas-phase sequencer equipped with an automatic on-line phenylthio\ufffehydantoin analyser. Preparation of apoenzyme and reconstruction of flavo\ufffeprotein. Eight millilitres of 25 mM potassium phosphate (pH 7?1) containing 1 M (NH4)2SO4, 1 M KBr and 1 mM EDTA was mixed with 2 ml purified enzyme (5 mg). The sample was applied to the HiPrep 16/10 Phenyl FF column equilibrated with the same buffer. The column was washed with 40 ml starting buffer. Flavin was eluted with 60 ml of the same buffer adjusted to pH 4?0 with 1 M phosphoric acid. After flavin elution, the column was immediately washed with 40 ml starting buffer, omitting 1 M KBr. Four hundred millilitres of a linear gradient of (NH4)2SO4 from 1 to 0 M in the same buffer was applied. This was followed by further elution with 100 ml starting buffer. The fractions (30 ml) containing apoenzyme were concentrated (Van Berkel et al., 1988). The protein was reconstructed directly on the column. After the release of the original flavin from the column, the pH of the column was equilibrated back to 7?1, as described above. Forty millilitres of the starting buffer containing 1 mM FMN, FAD, or riboflavin was passed through the column at a flow rate of 0?5 ml min21 . The column was placed at 4 uC overnight and then washed with 40 ml starting buffer, omitting 1 M KBr, to remove the unbound excess flavin. The elution of the protein was done as above (Van Berkel et al., 1988). Identification of Methyl Red and its metabolites. Two assay mixtures containing 0?4 mM NADPH and 50 mM Methyl Red in 20 ml of 25 mM sodium phosphate buffer (pH 7?1) with or without the purified enzyme were prepared. Methyl Red, N,N-dimethyl-p\ufffephenylenediamine and 2-aminobenzoic acid (Sigma) solutions (50 mg l21 ), using acetonitrile as solvent, were prepared. The assay mixture containing the enzyme was then incubated at room tem\ufffeperature until no decrease in optical density at 430 nm was noted. The two assay mixtures were extracted three times with equal volumes of ethyl acetate, subsequent to adjusting the pH to 3 with 1 M HCl. The extracts were evaporated in a rotary evaporator at 40 uC and the remaining trace solvent was removed by evaporation at room temperature overnight. Each residue was dissolved in 1 ml acetonitrile and filtered through a 0?2 mm pore-size syringe filter. Forty microlitres of the samples was analysed with a Hewlett Packard HPLC 1050 equipped with a model variable-wavelength detector (detection wavelength was 250 nm) and a reverse-phase Inertsil 5m ODS-2 column (4?66250 mm, MetaChem Technologies). The mobile phase was composed of 25 mM phosphate buffer (pH 3?0) and acetonitrile (4 : 6, v/v) with a flow rate of 0?5 ml min21 (Moutaouakkil et al., 2003; Nakanishi et al., 2001; Wong & Yuen, 1998). Enzyme characterization. Each data point was analysed three times with a standard deviation of less than 10 %. The effects of pH on the activity and the optimum pH of S. aureus Sa-Azo1 were determined at room temperature with the following buffers: 0?1 M sodium acetate (pH 4?2\u20135?4), sodium phosphate (pH 5?8\u20137?8) and HEPES/NaOH (pH 8?2 and 8?6). The effects of temperature on Azo1 were determined by assaying the enzyme at temperatures ranging from 10 to 65 uC. Thermostability was measured by incubat\ufffeing the enzyme samples in 25 mM potassium phosphate buffer, pH 7?1, for 1 h at temperatures from 30 to 70 uC. The initial veloci\ufffeties of the enzymic reaction were obtained by varying the concentra\ufffetions of one substrate, Methyl Red (from 0?005 to 0?04 mM) or NADPH (from 0?05 to 0?4 mM), while the concentration of the other substrate was kept constant (NADPH, 0?4 mM or Methyl Red, 0?04 mM). Apparent Km and Vmax values were obtained from Lineweaver\u2013Burk plots."
  },
  "12706351": {
    "bleu": 92.99327354260089,
    "meteor": 94.68491581922767,
    "rouge1_p": 0.9454545454545454,
    "rouge1_r": 0.9754204398447607,
    "rouge1_f": 0.9602037567653613,
    "rouge2_p": 0.9209535759096612,
    "rouge2_r": 0.9501618122977347,
    "rouge2_f": 0.9353297228416693,
    "rougeL_p": 0.9448275862068966,
    "rougeL_r": 0.9747736093143596,
    "rougeL_f": 0.9595670168736071,
    "cosine_similarity": 0.9064945916898008,
    "precision": 0.8896551724137931,
    "recall": 0.8958333333333334,
    "ner_f1": 0.8927335640138409,
    "ner_tp": 129,
    "ner_fp": 16,
    "ner_fn": 15,
    "bertscore_p": 0.9516017436981201,
    "bertscore_r": 0.9680375456809998,
    "bertscore_f1": 0.9599469304084778,
    "bertscore_scibert_error": "The expanded size of the tensor (2227) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2227].  Tensor sizes: [1, 512]",
    "len_gt": 9393,
    "len_jl": 9597,
    "GT": "Materials and methods Microorganism and growth conditions The E. agglomerans strain used in this work was isolated from dye-contaminated sludge collected from an industrial area in Casablanca (Morocco). Biochemical analysis according to the standardized micromethod API 20 E (bioMerieux, Inc.) allowed bacterial identification. This bacterial strain, which was found to have a higher ability to decolorize and degrade the toxic azo dye methyl red under aerobic conditions, was grown aerobically at 37 \u00b0C for 24 h in nutrient broth (Topley House, Bury, England) containing 100 mg/L of MR. The culture was inoculated with 1% (v/v) of overnight preculture in the same medium without MR. Crude extract preparation Cells from 2.5 L of culture were harvested by centrifugation at 9500g for 10 min, washed three times with 50 mM sodium phosphate buffer (pH 7.0), and suspended in 50 ml of the same buffer containing 0.5 mM EDTA and 0.1% (v/v) 2-mercaptoethanol (buffer A). Cells were disrupted in the cold by sonication (30 s, 70% output, 16) using a Bandelin Sonopuls sonifier. Cellular debris and unbroken cells were removed by centrifugation at 15,000g for 45 min at 4 \u00b0C using a Sigma 3K15 refrigerated centrifuge. The supernatant obtained constitutes the crude bacterial extract (soluble protein fraction). Purification procedure The enzyme was purified by a four-step procedure carried out at 4 \u00b0C. Ammonium sulfate precipitation. Crude extract was brought to 32% (w/v) saturation with solid ammonium sulfate ((NH\u2084)\u2082SO\u2084), stirred for 2 h, and then centrifuged at 15,000g for 45 min. Afterward, the resulting supernatant was precipitated with ammonium sulfate to a final saturation of 48% (w/v). The final pellet after centrifugation (45 min at 15,000g) was dissolved in a minimal volume of buffer A. The protein solution was dialyzed twice against 1 L of the same buffer overnight. Molecular exclusion chromatography. The dialyzed enzyme preparation was then applied to a Sephadex G-75 (Pharmacia Fine Chemicals, Uppsala, Sweden) column (1.6 \u00d7 60 cm) equilibrated with two bed volumes of buffer A. The enzyme was then eluted with equilibrating buffer at a flow rate of 10 ml/h. Fractions of 2 ml were collected and those that showed azoreductase activity were pooled. Ion-exchange chromatography. The enzyme preparation from above was applied at a flow rate of 6 ml/h to a DEAE\u2013cellulose (Serva, Heidelberg, Germany) column (3 \u00d7 12 cm) that had been previously equilibrated with buffer A. The column was extensively washed at the same flow rate with equilibrating buffer solution. Elution was performed with a linear gradient of sodium chloride (NaCl) (0\u2013500 mM; total volume of 200 ml) in buffer A. Fractions of 2 ml were collected and those which showed azoreductase activity were pooled and dialyzed twice against 1 L of buffer A overnight. Dye-affinity chromatography. The dialyzed enzyme preparation was then loaded onto a Cibacron blue\u2013agarose 3GA (Sigma, St. Louis, MO, USA) column (1 \u00d7 10 cm) equilibrated with two bed volumes of buffer A. The column was extensively washed at a flow rate of 20 ml/h and then eluted with equilibrating buffer containing 10 mM of NADH at a flow rate of 6 ml/h. The fractions with maximal activity were collected and pooled. Purified enzyme was made up to 50% glycerol and stored at \u221220 \u00b0C until use. Assay of azoreductase activity The activity of azoreductase was determined spectrophotometrically at 25 \u00b0C, using a Jenway 6405 UV/Visible spectrophotometer, by monitoring NADH disappearance at 340 nm based on the procedure described by Zimmermann et al. [16]. In general, enzyme preparation was added to 50 mM sodium phosphate buffer (pH 7.0) containing 0.350 mM NADH (Sigma) and 90 \u00b5M MR; the total volume of the reaction mixture was 1.0 ml. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 \u00b5mol of NADH/min. All experiments and assays were carried out in triplicate. Protein concentration Protein concentration was measured according to the Bradford [23] procedure, using bovine serum albumin (BSA) as a standard. Analytical gel electrophoresis Denaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS\u2013PAGE) was performed as described by Laemmli [24] on one-dimensional 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments). After electrophoresis, gels were stained with Coomassie brilliant blue R-250 at 0.025% (w/v) in methanol/acetic acid/water (4:1:5, v/v/v) for 30 min at room temperature. Destaining was done in methanol/acetic acid/water (4:1:5, v/v/v). The apparent subunit molecular weight was determined by measuring relative mobilities and comparing with the prestained SDS\u2013PAGE molecular weight standards (Precision Plus Protein Standards, Bio-Rad). Native molecular weight determination. To determine the native molecular weight of purified azoreductase, nondenaturing polyacrylamide gel electrophoresis was carried out according to the method of Hedrick and Smith [25]. The separating gels (6, 8, 10, and 12% polyacrylamide) were buffered with 1.5 M Tris\u2013HCl (pH 8.8). The running buffer was composed of 25 mM Tris and 320 mM glycine (pH 8.6). All experiments were realized at 4 \u00b0C. The electrophoresis running conditions, staining, and destaining were as described for SDS\u2013PAGE. The relative molecular weight of the native purified azoreductase was estimated using a commercial rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (137,000), BSA (66,000), ovalbumin (45,000), and trypsin (23,000) (Sigma) as molecular weight markers. By constructing the Ferguson plot [log(Rf \u00d7 100) versus the concentration of polyacrylamide gels (%)], the resulting slopes versus the standard native proteins of known molecular weight allows determination of the molecular weight of purified azoreductase. HPLC analysis of methyl red and its metabolites MR and its degradation products were identified by reverse-phase HPLC analysis with UV detection using the method of Wong and Yuen [26] which we modified according to our conditions. Two assay mixtures containing 0.350 mM NADH and 90 \u00b5M MR in 10 ml of 50 mM sodium phosphate buffer (pH 7.0) with or without the purified enzyme were prepared. The assay mixture containing the enzyme was then incubated at 20 \u00b0C until no azoreductase activity was noted. The two assay mixtures were extracted three times with equal volumes of dichloromethane (DCM). The DCM extracts were pooled and evaporated to 2 ml at 40 \u00b0C in a rotary evaporator and then transferred to a test tube. The remaining DCM was removed by evaporation at room temperature (20 \u00b1 0.5 \u00b0C) and placed in a hood overnight. The extracted residue was dissolved in 5 ml acetonitrile and filtered through a 0.2-\u00b5m nylon filter; 20 \u00b5l was analyzed by a Jasco HPLC system equipped with a Model 875 variable-wavelength detector (detection wavelength was 254 nm) and a reverse-phase C18 column (25 cm \u00d7 4 mm) packed with 5-\u00b5m particles. The mobile phase was composed of 25 mM phosphate buffer (pH 3.0) and acetonitrile (4:6, v/v) with a flow rate of 0.2 ml/min. The standards of methyl red (BDH Chemicals, England), N,N\u2032-dimethyl-p-phenylenediamine (DMPD) (Fluka, Switzerland), and 2-aminobenzoic acid (ABA) (Fluka, Switzerland) were injected for comparison. Determination of optimal pH and temperature of purified azoreductase The influence of pH on the azoreductase activity was studied over a wide range of pH (from 4.0 to 11.0) using a mixture of different buffers that have different pKa (Tris, 4-morpholineethanesulfonic acid, Hepes, sodium phosphate, and sodium acetate) adjusted to the same ionic strength as the standard reaction mixture. Thermal activation experiments were carried out by measuring the activity in 50 mM sodium phosphate buffer (pH 7.0) at temperature range from 5 to 70 \u00b0C using a thermostated cuvette holder connected to a refrigerated bath circulator. Thermal denaturation experiments were carried out by enzyme incubation in 50 mM sodium phosphate buffer (pH 7.0) over a temperature range from 5 to 70 \u00b0C. After 10 min of incubation, aliquots were cooled in an ice bath and the residual activity was determined at 20 \u00b0C as described above. Kinetic studies of purified azoreductase Initial velocities of the enzymatic reaction were performed by varying the concentration of one substrate, MR (from 0.005 to 0.040 mM) or NADH (from 0.037 to 0.300 mM), while the concentration of the other substrate was kept constant (NADH or MR). Values of the Michaelis constants (Km) and maximal velocity (Vmax) for the reduction of MR and the oxidation of NADH by the purified azoreductase were determined from Lineweaver\u2013Burk double-reciprocal plots. Effect of cations on azoreductase activity The purified enzyme was incubated in the presence of different concentrations of cations (from 0.025 to 10 mM). For each concentration, residual activity was measured in comparison with control (aliquot without cations). The I50 was then estimated according to the procedure described by Job et al. [27]. Data can be plotted as Vo/Vi versus concentration of inhibitor. After plotting data for several different cation concentrations, a straight line is drawn through the points and the cation concentration that corresponds to Vo/Vi = 2 is the I50 value.",
    "LLM": "Materials and methods Microorganism and growth conditions The E. agglomerans strain used in this work was isolated from dye-contaminated sludge collected from an industrial area in Casablanca (Morocco). Biochemi- cal analysis according to the standardized micromethod API 20 E (bioMerieux, Inc.) allowed bacterial identifi- cation. This bacterial strain, which was found to have a higher ability to decolorize and degrade the toxic azo dye methyl red under aerobic conditions, was grown aerobically at 37 \u0001C for 24 h in nutrient broth (Topley House, Bury, England) containing 100 mg/L of MR. The culture was inoculated with 1% (v/v) of overnight preculture in the same medium without MR. Crude extract preparation Cells from 2.5 L of culture were harvested by centri- fugation at 9500g for 10 min, washed three times with 50 mM sodium phosphate buffer (pH 7.0), and sus- pended in 50 ml of the same buffer containing 0.5 mM EDTA and 0.1% (v/v) 2-mercaptoethanol (buffer A). Cells were disrupted in the cold by sonication (30 s, 70% output, 16) using a Bandelin Sonopuls sonifier. Cel- lular debris and unbroken cells were removed by cen- trifugation at 15,000g for 45 min at 4 \u0001C using a Sigma 3K15 refrigerated centrifuge. The supernatant obtained constitutes the crude bacterial extract (soluble protein fraction). Purification procedure The enzyme was purified by a four-step procedure carried out at 4 \u0001C. Ammonium sulfate precipitation. Crude extract was brought to 32% (w/v) saturation with solid ammonium sulfate \u00f0\u00f0NH4\u00de2SO4\u00de, stirred for 2 h, and then centri- fuged at 15,000g for 45 min. Afterward, the resulting supernatant was precipitated with ammonium sulfate to a final saturation of 48% (w/v). The final pellet after centrifugation (45 min at 15,000g) was dissolved in a minimal volume of buffer A. The protein solution was dialyzed twice against 1 L of the same buffer overnight. Molecular exclusion chromatography. The dialyzed enzyme preparation was then applied to a Sephadex G- 75 (Pharmacia Fine Chemicals, Uppsala, Sweden) col- umn (1:6 60 cm) equilibrated with two bed volumes of buffer A. The enzyme was then eluted with equilibrating buffer at a flow rate of 10 ml/h. Fractions of 2 ml were collected and those that showed azoreductase activity were pooled. Ion-exchange chromatography. The enzyme prepara- tion from above was applied at a flow rate of 6 ml/h to a DEAE\u2013cellulose (Serva, Heidelberg, Germany) column (3 12 cm) that had been previously equilibrated with buffer A. The column was extensively washed at the same flow rate with equilibrating buffer solution. Elu- tion was performed with a linear gradient of sodium chloride (NaCl) (0\u2013500 mM; total volume of 200 ml) in buffer A. Fractions of 2 ml were collected and those which showed azoreductase activity were pooled and dialyzed twice against 1 L of buffer A overnight. Dye-affinity chromatography. The dialyzed enzyme preparation was then loaded onto a Cibacron blue\u2013 agarose 3GA (Sigma, St. Louis, MO, USA) column (1 10 cm) equilibrated with two bed volumes of buffer A. The column was extensively washed at a flow rate of 20 ml/h and then eluted with equilibrating buffer con- taining 10 mM of NADH at a flow rate of 6 ml/h. The fractions with maximal activity were collected and pooled. Purified enzyme was made up to 50% glycerol and stored at )20 \u0001C until use. Assay of azoreductase activity The activity of azoreductase was determined spec- trophotometrically at 25 \u0001C, using a Jenway 6405 UV/ Visible spectrophotometer, by monitoring NADH dis- appearance at 340 nm based on the procedure described by Zimmermann et al. [16]. In general, enzyme prepa- ration was added to 50 mM sodium phosphate buffer (pH 7.0) containing 0.350 mM NADH (Sigma) and 90 lM MR; the total volume of the reaction mixture was 1.0 ml. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 lmol of NADH/min. All experiments and assays were carried out in triplicate. Protein concentration Protein concentration was measured according to the Bradford [23] procedure, using bovine serum albumin (BSA) as a standard. 1 Abbreviations used: MR, methyl red; BSA, bovine serum albumin; DCM, dichloromethane; DMPD, N;N0 -dimethyl-p-phenylenediamine; ABA, 2-aminobenzoic acid. Analytical gel electrophoresis Denaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS\u2013PAGE) was performed as described by Laemmli [24] on one-dimensional 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments). After electrophoresis, gels were stained with Coomassie brilliant blue R-250 at 0.025% (w/v) in methanol/acetic acid/water (4:1:5, v/v/v) for 30 min at room temperature. Destaining was done in methanol/acetic acid/water (4:1:5, v/v/v). The apparent subunit molecular weight was determined by measuring relative mobilities and com- paring with the prestained SDS\u2013PAGE molecular weight standards (Precision Plus Protein Standards, Bio-Rad). Native molecular weight determination. To determine the native molecular weight of purified azoreductase, nondenaturing polyacrylamide gel electrophoresis was carried out according to the method of Hedrick and Smith [25]. The separating gels (6, 8, 10, and 12% polyacrylamide) were buffered with 1.5 M Tris\u2013HCl (pH 8.8). The running buffer was composed of 25 mM Tris and 320 mM glycine (pH 8.6). All experiments were re- alized at 4 \u0001C. The electrophoresis running conditions, staining, and destaining were as described for SDS\u2013 PAGE. The relative molecular weight of the native pu- rified azoreductase was estimated using a commercial rabbit muscle glyceraldehyde-3-phosphate dehydroge- nase (137,000), BSA (66,000), ovalbumin (45,000), and trypsin (23,000) (Sigma) as molecular weight markers. By constructing the Ferguson plot [log\u00f0Rf 100\u00de versus the concentration of polyacrylamide gels (%)], the re- sulting slopes versus the standard native proteins of known molecular weight allows determination of the molecular weight of purified azoreductase. HPLC analysis of methyl red and its metabolites MR and its degradation products were identified by reverse-phase HPLC analysis with UV detection using the method of Wong and Yuen [26] which we modified according to our conditions. Two assay mixtures con- taining 0.350 mM NADH and 90 lM MR in 10 ml of 50 mM sodium phosphate buffer (pH 7.0) with or without the purified enzyme were prepared. The assay mixture containing the enzyme was then incubated at 20 \u0001C until no azoreductase activity was noted. The two assay mixtures were extracted three times with equal volumes of dichloromethane (DCM). The DCM ex- tracts were pooled and evaporated to 2 ml at 40 \u0001C in a rotary evaporator and then transferred to a test tube. The remaining DCM was removed by evaporation at room temperature (20 \u0007 0:5\u0001C) and placed in a hood overnight. The extracted residue was dissolved in 5 ml acetonitrile and filtered through a 0.2-lm nylon filter; 20 ll was analyzed by a Jasco HPLC system equipped with a Model 875 variable-wavelength detector (detec- tion wavelength was 254 nm) and a reverse-phase C18 column (25 cm 4mm) packed with 5-lm particles. The mobile phase was composed of 25 mM phosphate buffer (pH 3.0) and acetonitrile (4:6, v/v) with a flow rate of 0.2 ml/min. The standards of methyl red (BDH Chemicals, England), N; N0 -dimethyl-p-phenylenediamine (DMPD) (Fluka, Switzerland), and 2-aminobenzoic acid (ABA) (Fluka, Switzerland) were injected for comparison. Determination of optimal pH and temperature of purified azoreductase The influence of pH on the azoreductase activity was studied over a wide range of pH (from 4.0 to 11.0) using a mixture of different buffers that have different pKa (Tris, 4-morpholineethanesulfonic acid, Hepes, sodium phosphate, and sodium acetate) adjusted to the same ionic strength as the standard reaction mixture. Thermal activation experiments were carried out by measuring the activity in 50 mM sodium phosphate buffer (pH 7.0) at temperature range from 5 to 70 \u0001C using a thermostated cuvette holder connected to a re- frigerated bath circulator. Thermal denaturation exper- iments were carried out by enzyme incubation in 50 mM sodium phosphate buffer (pH 7.0) over a temperature range from 5 to 70 \u0001C. After 10 min of incubation, aliquots were cooled in an ice bath and the residual activity was determined at 20 \u0001C as described above. Kinetic studies of purified azoreductase Initial velocities of the enzymatic reaction were per- formed by varying the concentration of one substrate, MR (from 0.005 to 0.040 mM) or NADH (from 0.037 to 0.300 mM), while the concentration of the other sub- strate was kept constant (NADH or MR). Values of the Michaelis constants (Km) and maximal velocity (Vmax) for the reduction of MR and the oxidation of NADH by the purified azoreductase were determined from Li- neweaver\u2013Burk double-reciprocal plots. Effect of cations on azoreductase activity The purified enzyme was incubated in the presence of different concentrations of cations (from 0.025 to 10 mM). For each concentration, residual activity was measured in comparison with control (aliquot without cations). The I50 was then estimated according to the procedure described by Job et al. [27]. Data can be plotted as Vo=Vi versus concentration of inhibitor. After plotting data for several different cation concentrations, a straight line is drawn through the points and the cation concen- tration that corresponds to Vo=Vi \u00bc 2 is the I50 value."
  },
  "22182443": {
    "bleu": 97.3993288590604,
    "meteor": 97.62581126338745,
    "rouge1_p": 0.976905311778291,
    "rouge1_r": 0.9828066914498141,
    "rouge1_f": 0.9798471160528145,
    "rouge2_p": 0.9602587800369686,
    "rouge2_r": 0.9660622966062297,
    "rouge2_f": 0.963151796060255,
    "rougeL_p": 0.976905311778291,
    "rougeL_r": 0.9828066914498141,
    "rougeL_f": 0.9798471160528145,
    "cosine_similarity": 0.9530917598232826,
    "precision": 0.9032258064516129,
    "recall": 0.9032258064516129,
    "ner_f1": 0.9032258064516129,
    "ner_tp": 140,
    "ner_fp": 15,
    "ner_fn": 15,
    "bertscore_p": 0.9768134355545044,
    "bertscore_r": 0.9878959655761719,
    "bertscore_f1": 0.9824100136756897,
    "bertscore_scibert_error": "The expanded size of the tensor (2914) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2914].  Tensor sizes: [1, 512]",
    "len_gt": 12930,
    "len_jl": 12938,
    "GT": "2. Materials and methods 2.1. Bacterial strains, Cultivation, Plasmids, and Azo dyes C. perfringens ATCC 3626 was grown on sheep blood agar plates in an anaerobic jar for 3 days and used to inoculate 10 ml of anaerobic brain heart infusion broth (BHI) in an anaerobic chamber. The broth culture was used for the preparation of chromosomal DNA and for inoculating additional tubes of 10 ml anaerobic BHI for assays. TOP10 cells (Invitrogen) were used for initial cloning. NovaBlueDE3 and BL21pLysS cells (Novagen) were used for expression. For initial cloning, the host cells were grown in Luria Bertani (LB) medium containing 50 mg/ml ampicillin. For protein expression, the host strain was grown in LB medium containing 50 mg/ml ampicillin and 12.5 mg/ml tetracycline. Plasmid pCR 2.1-TOPO (Invitrogen) was used for initial cloning. The pET-15b plasmid (Novagen) was used for protein expression. The azo dyes were purchased from the following companies: Direct Blue 15 (MP Biomedicals), Methyl Red (Acros Organics), Tartrazine (Sigma\u2013Aldrich), Trypan Blue (Kodak), Congo Red (Sigma\u2013Aldrich), Eriochrome Black T (MCB), Buffalo Black NBR (Allied Chemical), Janus Green (Sigma\u2013Aldrich) and Cibacron Brilliant Red 3B-A (Sigma\u2013Aldrich). 2.2. Reduction of Azo dyes by C. perfringens whole cells A volume of 100 ml fresh C. perfringens culture was used to inoculate 10 ml of anaerobic BHI under anaerobic conditions. Stock solutions of 1 mg/ml were prepared for the azo dyes Direct Blue 15, Methyl Red, and Tartrazine and added to each respective tube to a final concentration of 10 mM. Each assay consisted of 10 ml BHI containing 10 mM dye, 10 ml BHI inoculated with only C. perfringens, and 10 ml BHI inoculated with both 10 mM dye and C. perfringens and were performed in triplicate. The samples were kept at 37 \u00b0C without agitation. One milliliter samples were removed every 1.5 h for a minimum of 6 h with a sterile needle and syringe followed by centrifugation at 14,000 g for 1 min. The absorbance of Direct Blue 15, Methyl Red, or Tartrazine in the supernatant was measured spectrophotometrically at 606.5 nm, 430 nm, and 427 nm, respectively. The pellet was resuspended in 1 ml of sterile water and the absorbance was measured at 600 nm. The ability of C. perfringens cells to reduce higher concentrations of azo dye and the effect of these concentrations on cell growth was further tested in the presence of 20 mM and 40 mM Methyl Red. The ability of C. perfringens to reduce other azo dyes was tested in the presence of 10 mM concentrations of Janus Green, Congo Red, and Trypan Blue in BHI. The assays were similar to those just described, however, measurements were taken once and only after incubation for 12 h in the presence of each dye. 2.3. Identification of the azoC gene in C. perfringens TBLASTN and TBLASTP software (National Center for Toxicological Research) was used to search the C. perfringens chromosome for proteins showing homology to the deduced nucleotide and amino acid sequences of the azoreductases from E. coli, Enterococcus faecalis, Staphylococcus aureus and Bacillus OY1-2. A list of known bacterial azoreductases and their conserved domains was obtained from the Universal Protein Resource (UniProt) Knowledgebase. The Protein Family Database (PFAM) was used to identify proteins with these conserved domains within the C. perfringens chromosome and also to compare their function(s) to that of known azoreductases. 2.4. Cloning of the C. perfringens azoC ORF and expression of AzoC in E. coli Genomic DNA was extracted from a fresh 10 ml culture of C. perfringens ATCC 3626 according to the Masterpure Gram Positive DNA Purification Kit (Epicentre Biotechnologies). The azoC open reading frame was amplified using C. perfringens genomic DNA as template. The forward primer (flavoF3) included an XhoI site before the start codon: 5\u2032-CGGCCTCGAGATGAAAGTATTATTAGTTA-3\u2032. The reverse primer (flavo0915r) included a BamHI site downstream of the azoC ORF: 5\u2032-GTGAAAGGATCCTTATCTAATAAAATTAGTTCTTTCTCTTTC-3\u2032. Template DNA and primers for the control reaction were provided in the TOPO TA cloning kit (Invitrogen). PCR was carried out in a Perkin Elmer 480 DNA thermal cycler. Conditions for amplification were followed as outlined in the TOPO TA cloning manual (Invitrogen) - one cycle of 2 min at 94 \u00b0C, 25 cycles with each cycle consisting of 1 min at 94 \u00b0C, 1 min at 55 \u00b0C, and 1 min at 72 \u00b0C, and a final extension of 7 min at 72 \u00b0C. The products were analyzed on a 1% agarose gel after staining with ethidium bromide. The PCR product containing the azoC ORF was directly cloned into pCR 2.1-TOPO TA vector (Invitrogen) and sequenced. The TOPO clone and pET15b (Novagen) were each cleaved by sequential digestion with XhoI and BamHI restriction enzymes, each time at 37 \u00b0C for 12 h. The 721 bp product from cleavage of the TOPO clone was purified using the QIAquick Gel Extraction Kit (QIAGEN) and ligated between the XhoI and BamHI sites of pET15b in a reaction containing 0.1 U T4 DNA ligase and carried out at 4 \u00b0C for 16 h. A 2 ml volume of the ligation mixture was used to transform 20 ml of NovaBlueDE3 cells (Novagen). Following plasmid extractions, positive clones were confirmed by PCR with the insert specific primers flavoF3 and flavo0915r and by simultaneous cleavage with XhoI and BamHI. The recombinant DNA (pAzoC) was used to transform BL21pLysS cells, which were deficient in protease activity, and used for expression of the azoreductase. BL21pLysS cells harboring pAzoC were grown at 37 \u00b0C overnight in 300 ml of LB broth containing 50 mg/ml ampicillin and 34 mg/ml chloramphenicol. BL21pLysS lacking the plasmid was cultured under the same conditions in the presence of 34 mg/ml chloramphenicol and used as a control. The cultures were grown and the total protein isolated as described in Macwana et al. [20]. The concentration of total protein (mg/ml) was determined using a Nanodrop spectrophotometer. The samples were analyzed by SDS-PAGE to confirm molecular weight and expression levels. 2.5. Purification of the enzyme The NovaBlue (DE3) E. coli cells containing the pAzoC and His-tag as prepared above were used to inoculate LB broth and protein was grown and isolated as described in Macwana et al. [20]. The protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) slurry (ClonTech) in a 1:1 ratio of supernatant to slurry. Purification also followed the procedure detailed in Macwana et al. [20]. All purification elutions were analyzed with SDS-PAGE gel and protein concentration was determined with a Nanodrop spectrometer, using the Molar Extinction Coefficient of the protein (0.880) to determine the protein concentration. The Molar Extinction Coefficient was determined by using the NCBI database. All samples were concentrated using an Amicon Ultrafiltration cell under Nitrogen pressure and a Millipore filter (Regenerated Cellulose membrane, NMWL 10,000) to achieve a suitable protein concentration of between 2 and 5 mg/ml. 2.6. Pure enzyme assays under anaerobic and aerobic conditions Assays for azoreductase activity for the pure AzoC protein were carried out both anaerobically and aerobically. All experiments were performed in triplicate. Enzyme reactions were carried out in a 1.5 ml-polystyrene cuvette (Sigma\u2013Aldrich) with a total reaction volume of 1 ml. The buffer used in most cases (except for specific pH experiments) was 25 mM TRIS, pH 9.0. Although different azo dyes were tested the final concentration tested was consistently 20 mM. Experiments were performed to determine the optimal enzyme concentration in the reaction by altering the concentration of the protein and once the optimal AzoC concentration was found, different cofactors were tested (NADH and NADPH). The concentration of NADH (Acros Organics) was also altered in experiments to determine optimal conditions for dye reduction (10 mM NADH). FAD (Sigma\u2013Aldrich) and FMN (Sigma\u2013Aldrich) were both tested as cofactors and FAD was found to be an essential component to the azoreductase function of AzoC at an optimal concentration of 2 mM. Reactions were prepared by mixing the appropriate buffer, azo dye, water and the enzyme together in the cuvette and bubbling with nitrogen at a rate of 1 nitrogen bubble per second for 10 min. Immediately following the nitrogen bubbling, mineral oil was added to the top of the cuvette to prevent the introduction of oxygen into the newly anaerobic system. Aerobic experiments were performed without the nitrogen bubbling step and without the addition of mineral oil. To begin the reaction, 10 mM NADH and 2 mM FAD were mixed together separately and then added to the reaction and mixed together carefully as to not introduce oxygen into the system. The reaction was scanned using a Shimadzu UV-1650 PC spectrophotometer. Each azo dye was scanned prior to the reaction with the spectrophotometer to determine the optimal absorbance for each dye (all dye absorption values were above 340 nm and extinction coefficients were used in determining the final concentration of dyes). In some cases, an absorbance was selected that was not the optimal due to interference by the absorbance of FAD, which is an orange color. The absorbencies tested were as follows: Direct Blue 15 (602.5 nm), Cibacron Brilliant Red 3B-A (534.00 nm), Congo Red (531.00 nm), Tartrazine (425.00 nm), Janus Green (597.50 nm), Trypan Blue (533.50 nm), Buffalo Black NBR (614.50 nm), Methyl Red (430.00 nm) and Eriochrome Black T (527.50 nm). Dye concentration was interpolated from absorbencies by making standard curves of dye concentrations to find the extinction coefficient. This was determined to be a spectrophotometer-specific equation of a best fit line. Experiments were also done to determine the optimal order of the addition of reactants to the system where each of the components (dye, AzoC, cofactors) were added last in separate experiments. 2.7. Pure enzyme assays \u2013 cofactor effect To determine the effect of the cofactor on the enzyme reaction, experiments were performed that lacked the azoreductase and contained a protein control, albumin (Sigma\u2013Aldrich). The protein control would serve to show whether the azoreductase was causing the reduction of the dye or if the cofactors and the presence of a protein was causing the reduction. Experiments were also performed that did not contain a protein and showed the effects of the cofactors themselves. In the case of the protein control, 138 \u00b5g of albumin was added to the reaction in place of the AzoC. For the cofactor controls, only NADH and FAD were added to the reaction; no protein was present in these experiments. 2.8. Pure enzyme assays \u2013 optimal temperature To determine the optimal temperature for the azoreductase enzyme to work, experiments were carried out at several different temperatures (25 \u00b0C/room temperature, 30 \u00b0C, 37 \u00b0C and 45 \u00b0C). Prior to the reaction taking place, all components except for the cofactors were incubated in their cuvette in a water bath to reach the desired temperature. The cuvette was moved to the spectrophotometer to begin the experiment with the cofactors and then promptly moved back to the water bath. The absorbance was recorded every 5 min until the reading reached a plateau. 2.9. Pure enzyme assays \u2013 optimal pH To determine the optimal pH for AzoC to work, several different pH buffers were prepared to test azoreductase activity: Sodium acetate pH 4.0 and 5.0, Potassium Phosphate pH 6.0 and 7.0, TRIS pH 8.0 and 9.0, Sodium bicarbonate pH 10.0 and 11.0, and Potassium chloride pH 12.0. Enzyme experiments were conducted as described above by only changing the pH of the buffer used. 2.10. Pure enzyme assays \u2013 effect of oxygen Since AzoC is from an anaerobic organism, there was curiosity as to the effect of oxygen on the protein itself. To study this, anaerobic and aerobic experiments were performed and compared as mentioned above. In another experiment, an anaerobic reaction at the optimal conditions was prepared and taken to complete dye reduction. Following dye reduction, the cuvette was bubbled again (in one case with Nitrogen and in another case with Oxygen). Dye and cofactors were added into the reaction and the cuvette scanned. This provides an understanding of the regeneration of the enzyme in the presence of oxygen as compared to anaerobically. 2.11. Pure enzyme assays \u2013 enzyme kinetics A brief study of the enzyme kinetics was performed by taking one component of the experiment (dye, FAD, NADH) and changing the concentration of that component while keeping the others constant. The optimal conditions were 20 mM dye, 10 mM NADH and 2 mM FAD. During the kinetics experiments, the optimal concentration of one of these components was tested by either increasing or decreasing in hopes of finding a saturation point for the enzyme. Results were graphed and analyzed by a Lineweaver\u2013Burk plot to determine Km and Vmax.",
    "LLM": "2. Materials and methods 2.1. Bacterial strains, Cultivation, Plasmids, and Azo dyes C. perfringens ATCC 3626 was grown on sheep blood agar plates in an anaerobic jar for 3 days and used to inoculate 10 ml of anaerobic brain heart infusion broth (BHI) in an anaerobic chamber. The broth culture was used for the preparation of chromosomal DNA and for inoculating additional tubes of 10 ml anaerobic BHI for assays. TOP10 cells (Invitrogen) were used for initial cloning. NovaBlueDE3 and BL21pLysS cells (Novagen) were used for expression. For initial cloning, the host cells were grown in Luria Bertani (LB) medium containing 50 mg/ml ampicillin. For protein expression, the host strain was grown in LB medium containing 50 mg/ml ampicillin and 12.5 mg/ml tetracycline. Plasmid pCR 2.1- TOPO (Invitrogen) was used for initial cloning. The pET-15b plasmid (Novagen) was used for protein expression. The azo dyes were purchased from the following companies: Direct Blue 15 (MP Biomedicals), Methyl Red (Acros Organics), Tartrazine (Sigma\ufffeeAldrich), Trypan Blue (Kodak), Congo Red (SigmaeAldrich), Eriochrome Black T (MCB), Buffalo Black NBR (Allied Chemical), Janus Green (SigmaeAldrich) and Cibacron Brilliant Red 3B-A (SigmaeAldrich). 2.2. Reduction of Azo dyes by C. perfringens whole cells A volume of 100 ml fresh C. perfringens culture was used to inoculate 10 ml of anaerobic BHI under anaerobic conditions. Stock solutions of 1 mg/ml were prepared for the azo dyes Direct Blue 15, Methyl Red, and Tartrazine and added to each respective tube to a final concentration of 10 mM. Each assay consisted of 10 ml BHI containing 10 mM dye, 10 ml BHI inoculated with only C. perfringens, and 10 ml BHI inoculated with both 10 mM dye and C. perfringens and were performed in triplicate. The samples were kept at 37 C without agitation. One milliliter samples were removed every 1.5 h for a minimum of 6 h with a sterile needle and syringe followed by centrifugation at 14,000 g for 1 min. The absorbance of Direct Blue 15, Methyl Red, or Tartrazine in the supernatant was measured spectrophotometrically at 606.5 nm, 430 nm, and 427 nm, respectively. The pellet was resuspended in 1 ml of sterile water and the absorbance was measured at 600 nm. The ability of C. perfringens cells to reduce higher concentrations of azo dye and the effect of these concentrations on cell growth was further tested in the presence of 20 mM and 40 mM Methyl Red. The ability of C. perfringens to reduce other azo dyes was tested in the presence of 10 mM concentrations of Janus Green, Congo Red, and Trypan Blue in BHI. The assays were similar to those just described, however, measurements were taken once and only after incubation for 12 h in the presence of each dye. 2.3. Identification of the azoC gene in C. perfringens TBLASTN and TBLASTP software (National Center for Toxico\ufffelogical Research) was used to search the C. perfringens chromosome for proteins showing homology to the deduced nucleotide and amino acid sequences of the azoreductases from E. coli, Entero\ufffecoccus faecalis, Staphylococcus aureus and Bacillus OY1-2. A list of known bacterial azoreductases and their conserved domains was obtained from the Universal Protein Resource (UniProt) Knowl\ufffeedgebase. The Protein Family Database (PFAM) was used to identify proteins with these conserved domains within the C. perfringens chromosome and also to compare their function(s) to that of known azoreductases. 2.4. Cloning of the C. perfringens azoC ORF and expression of AzoC in E. coli Genomic DNA was extracted from a fresh 10 ml culture of C. per\ufffefringens ATCC 3626 according to the Masterpure Gram Positive DNA Purification Kit (Epicentre Biotechnologies). The azoC open reading frame was amplified using C. perfringens genomic DNA as template. The forward primer (flavoF3) included an Xho1 site before the start codon: 50 - CGGCCTCGAGATGAAAGTATTATTAGTTA e 30 . The reverse primer (flavo0915r) included a BamH1 site downstream of the azoC ORF: 50 GTGAAAGGATCCTTATCTAATAAAATTAGTTCTTTCTCTTTC 30 . Template DNA and primers for the control reaction were provided in the TOPO TA cloning kit (Invitrogen). PCR was carried out in a Perkin Elmer 480 DNA thermal cycler. Conditions for amplification were followed as outlined in the TOPO TA cloning manual (Invitrogen) - one cycle of 2 min at 94 C, 25 cycles with each cycle consisting of 1 min at 94 C, 1 min at 55 C, and 1 min at 72 C, and a final extension of 7 min at 72 C. The products were analyzed on a 1% agarose gel after staining with ethidium bromide. The PCR product containing the azoC ORF was directly cloned into pCR 2.1-TOPO TA vector (Invitrogen) and sequenced. The TOPO clone and pET15b (Novagen) were each cleaved by sequential digestion with XhoI and BamHI restriction enzymes, each time at 37 C for 12 h. The 721 bp product from cleavage of the TOPO clone was purified using the QIAquick Gel Extraction Kit (QIAGEN) and ligated between the XhoI and BamHI sites of pET15b in a reaction containing 0.1U T4 DNA ligase and carried out at 4 C for 16 h. A 2 ml volume of the ligation mixture was used to transform 20 ml of NovaBlueDE3 cells (Novagen). Following plasmid extractions, positive clones were confirmed by PCR with the insert specific primers flavoF3 and flavo0915r and by simultaneous cleavage with XhoI and BamHI. The recombinant DNA (pAzoC) was used to transform BL21pLysS cells, which were deficient in protease activity, and used for expression of the azoreductase. BL21pLysS cells harboring pAzoC were grown at 37 C overnight in 300 ml of LB broth containing 50 mg/ml ampicillin and 34 mg/ml chloramphenicol. BL21pLysS lacking the plasmid was cultured under the same conditions in the presence of 34 mg/ml chloram\ufffephenicol and used as a control. The cultures were grown and the total protein isolated as described in Macwana et al [20]. The concentration of total protein (mg/ml) was determined using a Nanodrop spectrophotometer. The samples were analyzed by SDS-PAGE to confirmed molecular weight and expression levels. 2.5. Purification of the enzyme The NovaBlue (DE3) E. Coli cells containing the pAzoC and His-tag as prepared above were used to inoculate LB broth and protein was grown and isolated as described in Macwana et al [20]. The protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) slurry (Clon\ufffeTech) in a 1:1 ratio of supernatant to slurry. Purification also followed the procedure detailed in Macwana et al[20]. All purification elutions were analyzed with SDS-PAGE gel and protein concentration was determined with a Nanodrop spectrometer, using the Molar Extinc\ufffetion Coefficient of the protein (0.880) to determine the protein concentration. The Molar Extinction Coefficient was determined by using the NCBI database. All samples were concentrated using an Amicon Ultrafiltration cell under Nitrogen pressure and a Millipore filter (Regenerated Cellulose membrane, NMWL 10,000) to achieve a suitable protein concentration of between 2 and 5 mg/mL. 2.6. Pure enzyme assays under anaerobic and aerobic conditions Assays for azoreductase activity for the pure AzoC protein were carried out both anaerobically and aerobically. All experiments were performed in triplicate. Enzyme reactions were carried out in a 1.5 mL-polystyrene cuvette (SigmaeAldrich) with a total reaction volume of 1 mL. The buffer used in most cases (except for specific pH experiments) was 25 mM TRIS, pH 9.0. Although different azo dyes were tested the final concentration tested was consistently 20 mM. Experiments were performed to determine the optimal enzyme concentration in the reaction by altering the concentration of the protein and once the optimal AzoC concentration was found, different cofactors were tested (NADH and NADPH). The concen\ufffetration of NADH (Acros Organics) was also altered in experiments to determine optimal conditions for dye reduction (10 mM NADH). FAD (SigmaeAldrich) and FMN (SigmaeAldrich) were both tested as cofactors and FAD was found to be an essential component to the azoreductase function of AzoC at an optimal concentration of 2 mM. Reactions were prepared by mixing the appropriate buffer, azo dye, water and the enzyme together in the cuvette and bubbling with nitrogen at a rate of 1 nitrogen bubble per second for 10 min. Immediately following the nitrogen bubbling, mineral oil was added to the top of the cuvette to prevent the introduction of oxygen into the newly anaerobic system. Aerobic experiments were performed without the nitrogen bubbling step and without the addition of mineral oil. To begin the reaction, 10 mM NADH and 2 mM FAD were mixed together separately and then added to the reaction and mixed together carefully as to not introduce oxygen into the system. The reaction was scanned using a Shimadzu UV\ufffe1650 PC spectrophotometer. Each azo dye was scanned prior to the reaction with the spectrophotometer to determine the optimal absorbance for each dye (all dye absorption values were above 340 nm and extinction coefficients were used in determining the final concentration of dyes). In some cases, an absorbance was selected that was not the optimal due to interference by the absorbance of FAD, which is an orange color. The absorbencies tested were as follows: Direct Blue 15 (602.5 nm), Cibacron Brilliant Red 3B-A (534.00 nm), Congo Red (531.00 nm), Tartrazine (425.00 nm), Janus Green (597.50 nm), Trypan Blue (533.50 nm), Buffalo Black NBR (614.50 nm), Methyl Red (430.00 nm) and Erio\ufffechrome Black T (527.50 nm). Dye concentration was interpolated from absorbencies by making standard curves of dye concentra\ufffetions to find the extinction coefficient. This was determined to be a spectrophotometer-specific equation of a best fit line. Experi\ufffements were also done to determine the optimal order of the addition of reactants to the system where each of the components (dye, AzoC, cofactors) were added last in separate experiments. 2.7. Pure enzyme assays e cofactor effect To determine the effect of the cofactor on the enzyme reaction, experiments were performed that lacked the azoreductase and contained a protein control, albumin (SigmaeAldrich). The protein control would serve to show whether the azoreductase was causing the reduction of the dye or if the cofactors and the presence of a protein was causing the reduction. Experiments were also per\ufffeformed that did not contain a protein and showed the effects of the cofactors themselves. In the case of the protein control, 138 mg of albumin was added to the reaction in place of the AzoC. For the cofactor controls, only NADH and FAD were added to the reaction; no protein was present in these experiments. 2.8. Pure enzyme assays e optimal temperature To determine the optimal temperature for the azoreductase enzyme to work, experiments were carried out at several different temperatures (25 C/room temperature, 30 C, 37 C and 45 C). Prior to the reaction taking place, all components except for the cofactors were incubated in their cuvette in a water bath to reach the desired temperature. The cuvette was moved to the spectro\ufffephotometer to begin the experiment with the cofactors and then promptly moved back to the water bath. The absorbance was recorded every 5 min until the reading reached a plateau. 2.9. Pure enzyme assays e optimal pH To determine the optimal pH for AzoC to work, several different pH buffers were prepared to test azoreductase activity: Sodium acetate pH 4.0 and 5.0, Potassium Phosphate pH 6.0 and 7.0, TRIS pH 8.0 and 9.0, Sodium bicarbonate pH 10.0 and 11.0, and Potas\ufffesium chloride pH 12.0. Enzyme experiments were conducted as described above by only changing the pH of the buffer used. 2.10. Pure enzyme assays e effect of oxygen Since AzoC is from an anaerobic organism, there was curiosity as to the effect of oxygen on the protein itself. To study this, anaerobic and aerobic experiments were performed and compared as mentioned above. In another experiment, an anaerobic reaction at the optimal conditions was prepared and taken to complete dye reduction. Following dye reduction, the cuvette was bubbled again (in one case with Nitrogen and in another case with Oxygen). Dye and cofactors were added into the reaction and the cuvette scan\ufffened. This provides an understanding of the regeneration of the enzyme in the presence of oxygen as compared to anaerobically. 2.11. Pure enzyme assays e enzyme kinetics A brief study of the enzyme kinetics was performed by taking one component of the experiment (dye, FAD, NADH) and changing the concentration of that component while keeping the others constant. The optimal conditions were 20 mM dye, 10 mM NADH and 2 mM FAD. During the kinetics experiments, the optimal concentration of one of these components was tested by either increasing or decreasing in hopes of finding a saturation point for the enzyme. Results were graphed and analyzed by a Lineweaver\ufffeBurke plot to determine Km and Vmax."
  },
  "11134015": {
    "bleu": 99.57252143720761,
    "meteor": 99.58668679785913,
    "rouge1_p": 0.9971064814814815,
    "rouge1_r": 0.9982618771726536,
    "rouge1_f": 0.9976838448176029,
    "rouge2_p": 0.9936305732484076,
    "rouge2_r": 0.9947826086956522,
    "rouge2_f": 0.9942062572421784,
    "rougeL_p": 0.9965277777777778,
    "rougeL_r": 0.9976825028968713,
    "rougeL_f": 0.9971048060220035,
    "cosine_similarity": 0.9960623864133954,
    "precision": 0.9736842105263158,
    "recall": 0.9866666666666667,
    "ner_f1": 0.9801324503311258,
    "ner_tp": 148,
    "ner_fp": 4,
    "ner_fn": 2,
    "bertscore_p": 0.9985241293907166,
    "bertscore_r": 0.9979023337364197,
    "bertscore_f1": 0.9982205033302307,
    "bertscore_scibert_error": "The expanded size of the tensor (2474) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2474].  Tensor sizes: [1, 512]",
    "len_gt": 9905,
    "len_jl": 9904,
    "GT": "EXPERIMENTAL PROCEDURES Bacterial Strains, Plasmids, and Culture Conditions\u2014Azo dye-degrading bacteria isolated from soil near a waste-water plant from a textile factory were identified as Bacillus sp. OY1-2 based on biological characterization (9). This strain can grow in brain-heart infusion broth (Difco). E. coli strains C600hfl and XLI-Blue were cultured in Luria broth consisting of 10 g of bactotryptone (Difco), 5 g of yeast extract (Difco), and 10 g of NaCl per liter. The E. coli strains GI724 and GI618 were cultured in RMG medium consisting of 40 g of cazamino acids (Difco), 5 g of glycerol, 1 mM MgCl2, 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl per liter. Recombinant proteins were expressed by E. coli strains GI724 or GI618 in ID medium consisting of 0.4 g of cazamino acid, 5 g of glucose, 1 mM MgCl2, 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl per liter. A phage vector lgt10 was used for the construction of the genomic library. The plasmid pT7Blue(R)T (Novagen, Inc.) and pUC18 were used for the subcloning of genes. The plasmids pTrx-Fus (Invitrogen Co.) and pTrcNd (reconstructed from pQE30 (Qiagen Inc.) were used for expression of recombinant azoreductase. N-terminal Amino Acid Sequence of Native and Recombinant Azoreductase\u2014Samples of native and recombinant azoreductase were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blotted protein strips were used for amino acid sequencing on a PE Biosystems 470/120A protein sequencer. Construction of Bacillus sp. OY1-2 Genomic DNA Library\u2014Bacillus sp. OY1-2 genomic DNA was prepared by mechanical disruption as described previously (11). Briefly, bacterial pellet from 5 ml of liquid culture was suspended in 0.5 ml of lysis buffer consisting of 0.3 M Tris-HCl, pH 8.0, 0.1 M NaCl, and 6 mM EDTA. The cell suspension was transferred into a conical 2-ml screw-cap vial, which is one-fourth filled with 0.17-mm acid-washed sterile glass beads. Cells were disrupted by vigorous shaking with 0.5 ml of chloroform on a Mini-Bead Beater cell disrupter (Biospec Products, Bartlesville, UK) for 5 min. DNA in the upper layer after centrifugation was further purified by phenol/chloroform extraction, concentrated by ethanol precipitation, and dissolved in 300 ml of TE buffer consisting of 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA. The purified genomic DNA was completely digested with EcoRI and ligated into the EcoRI site of lgt10. Genomic library constructs were introduced into E. coli strain C600hfl by means of in vitro packaging using gigapack plus (Stratagene). Generation of Probe for Screening\u2014The N-terminal amino acid sequence was used to design oligonucleotide primers for amplifying the DNA fragment encoding the N-terminal of azoreductase (Fig. 2A). The reaction mixture (50 ml) consisted of long and accurate (LA) PCR buffer II (Mg21-free); 2.5 mM MgCl2; 200 mM each dATP, dCTP, dGTP, and dTTP; 10 ng of DNA from Bacillus sp. OY2-1; 1.25 units of Takara LA Taq DNA polymerase (Takara Shuzo Co., Ltd., Japan); and 0.5 mM each primer AZR-1 and AZR-2 (Fig. 2A). PCR was carried out for 30 cycles in a Takara Thermal Cycler Personal (Takara Shuzo Co., Ltd.), with each cycle consisting of denaturation for 30 s at 94 \u00b0C, annealing for 30 s at 55 \u00b0C, and extension for 1 min at 72 \u00b0C. The PCR product was extracted from the gel after separation on a 1% agarose gel and was directly subcloned into the pT7Blue(R)T vector. The subclones were sequenced by the dideoxy chain termination method (12) with a Model 310 genetic analyzer (PE Biochemicals Inc). A hybridization probe was synthesized by PCR using a PCR DIG labeling kit (Roche Diagnostics Co., Germany). The reaction mixture (50 ml) consisted of 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 mM each dATP, dCTP, and dGTP and 130 mM dTTP; 70 mM digoxigenin-11-dUTP; 1.25 units of Taq DNA polymerase; a 1 mM concentration of primers M13 M1 & M13 RV (Takara Shuzo Co., Ltd.); and 10 ng of plasmid carrying the PCR product encoding the N-terminal of azoreductase. PCR was performed under the same conditions as described above. Cloning and DNA Sequencing of the Azoreductase Gene\u2014A phage library was screened essentially as previously described (13). Approximately 1 \u00d7 105 plaques from the genomic library were plated with E. coli C600hfl and incubated at 37 \u00b0C for 6 h. Nylon filters (Nytran 13N, Schleicher & Schuell Co.) were processed for hybridization. The filters were prehybridized in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.) at 68 \u00b0C for 30 min and then hybridized for 1 h at 48 \u00b0C with a 10 ng/ml concentration of digoxigenin-labeled probe described above in the same buffer as used for prehybridization. The filters were washed for 5 min in 23 SSC and 0.1% SDS at room temperature followed by washing for 15 min in 0.23 SSC and 0.1% SDS at 48 \u00b0C. The hybridized probe was detected after 30 min of incubation at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antibody (Fab; Roche Diagnostics Co.) diluted 1:5000. The enzyme-catalyzed color reaction was carried out using a nitro blue tetrazolium salt (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) system (Wako Pure Chemical Industries, Japan) in Buffer 3 consisting of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2. The DNA inserts in the positive clones were subcloned into the EcoRI site of pUC18 for further characterization. The EcoRI fragment in the subclone was digested by SphI, NlaIV, or HincII; further subcloned in pUC18; and sequenced as shown in Fig. 2 by the dideoxy chain termination method with a Model 310 genetic analyzer. Database Search\u2014Protein and DNA sequences with homology to the deduced amino acid sequence of the azoreductase ORF were searched using TBLASTN from the National Center for Biological Information. Southern Blot Hybridization\u2014Detection of the restriction DNA fragment carrying the azoreductase gene was performed according to Southern (14). One mg of genomic DNA was completely digested with restriction enzymes, separated on a 0.7% agarose gel, and vacuum-transferred to Nytran 13N nylon filters. The filters were prehybridized in ExpressHyb hybridization solution at 68 \u00b0C for 30 min followed by hybridization with the same solution containing a 10 ng/ml Dig-labeled 1.2-kbp EcoRI DNA fragment carrying the whole coding region of azoreductase. After hybridization, the filters were washed for 5 min with 23 SSC and 0.1% SDS at room temperature followed by washing twice with 23 SSC/0.1% SDS for 15 min at 68 \u00b0C. The hybridized Dig-labeled probe on the filters were detected by alkaline phosphatase-conjugated anti-digoxigenin antibody, followed by color development using NBT/BCIP as substrates in Buffer 3. Expression of Azoreductase in E. coli\u2014The entire open reading frame of azoreductase was amplified by PCR. Briefly, the reaction mixture (50 ml) consisted of LA-PCR buffer II (Mg21-free); 2.5 mM MgCl2; 200 mM each dATP, dCTP, dGTP, and dTTP; 10 ng plasmid pT7B-AZR5\u20138; 1.25 units of Takara LA Taq DNA polymerase, and 0.5 mM each of primers AZR-rec-S-Nde (CATATGAAACTAGTCGTTATTAAC) and AZR-rec-E-Xba (TCTAGAGCAGATAGACTATTGGCTCC). PCR was carried out for 30 cycles in a Takara Thermal Cycler Personal, with each cycle consisting of denaturation for 30 s at 94 \u00b0C, annealing for 30 s at 55 \u00b0C, and extension for 1 min at 72 \u00b0C. The PCR product was extracted from the gel after separation on 1% agarose gel electrophoresis and subcloned into pT7Blue(R)T for confirmation of the nucleotide sequence and then transferred into expression vectors pTrx-Fus or pTrcNd after digestion by NdeI and XbaI. Expression of reductase in pTrx-Fus system was performed by adding tryptophan at a concentration of 0.1 mg/ml in ID medium. Expression in pTrcNd was performed by adding isopropyl-b-D-thio-galactopyranoside (IPTG) at a concentration of 1 mM in Luria-Bertani medium. Cells from 10 ml of induced culture were suspended in 0.5 ml of 20 mM sodium phosphate buffer, pH 7.0, lysed by two cycles of freezing at -80 \u00b0C and thawed at 37 \u00b0C followed by sonication (15 s, 70% output, 103). The supernatants from a 9000 \u00d7 g, 30 min centrifugation were used directly for enzyme assay or SDS-PAGE analysis. Purification of Recombinant Azoreductase by Red-Sepharose CL-6B\u2014The cells from 200 ml of culture were suspended in 20 ml of 20 mM sodium-phosphate buffer, pH 7.0 and lysed by freezing and thawing followed by sonication (15 s, 70% output, 103). After centrifugation at 9000 \u00d7 g for 30 min, the supernatant was applied to a Red-Sepharose CL-6B column (Amersham Pharmacia Biotech) followed by washing with 20 mM sodium phosphate buffer, pH 7.0. The recombinant azoreductase was eluted from the column with 10 mM b-NADH. The eluate was dialyzed against two changes of 1000 volumes of 20 mM sodium phosphate buffer, pH 7.0 and used for enzyme assay. Enzyme Assays\u2014Azo dye-reducing activity was analyzed by measuring the decrease in optical density at suitable wavelengths with a Hitachi U 3300 spectrophotometer at various temperatures basically according to Pasti-Grigsby et al. (15). The reaction mixture in a total volume of 1.0 ml consisted of various concentrations of azo dyes (Roccelin, Solar Orange, Sumifix Black B; shown in Fig. 3) in 20 mM sodium phosphate buffer, pH 7.0, and bacterial lysates or purified enzyme. The reaction mixture was preincubated for 5 min at the assay temperature, and the reaction was started by the addition of 25 ml of various concentrations of b-NADPH. The enzymatic activities were measured by the decrease in optical density at optimal wavelengths. The enzyme activity was expressed as the amount of reduced dye per min with 1 mg of enzyme. Kinetic parameters for the reduction of each dye by native and recombinant azoreductases were estimated by nonlinear regression analysis according to Shimada et al. (16).",
    "LLM": "EXPERIMENTAL PROCEDURES Bacterial Strains, Plasmids, and Culture Conditions\u2014Azo dye-degrading bacteria isolated from soil near a waste-water plant from a textile factory were identified as Bacillus sp. OY1-2 based on biological characterization (9). This strain can grow in brain-heart infusion broth (Difco). E. coli strains C600hfl and XLI-Blue were cultured in Luria broth consisting of 10 g of bactotryptone (Difco), 5 g of yeast extract (Difco), and 10 g of NaCl per liter. The E. coli strains GI724 and GI618 were cultured in RMG medium consisting of 40 g of cazamino acids (Difco), 5 g of glycerol, 1 mM MgCl2, 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl per liter. Recombinant proteins were expressed by E. coli strains GI724 or GI618 in ID medium consisting of 0.4 g of cazamino acid, 5 g of glucose, 1 mM MgCl2, 6 g of Na2HPO4,3g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl per liter. A phage vector lgt10 was used for the construction of the genomic library. The plasmid pT7Blue(R)T (Novagen, Inc.) and pUC18 were used for the subcloning of genes. The plasmids pTrx-Fus (Invitrogen Co.) and pTrcNd (reconstructed from pQE30 (Qiagen Inc.) were used for expression of recombinant azoreductase. N-terminal Amino Acid Sequence of Native and Recombinant Azoreductase\u2014Samples of native and recombinant azoreductase were separated on SDS-PAGE2 and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blotted protein strips were used for amino acid sequencing on a PE Biosystems 470/120A protein sequencer. Construction of Bacillus sp. OY1-2 Genomic DNA Library\u2014Bacillus sp. OY1-2 genomic DNA was prepared by mechanical disruption as described previously (11). Briefly, bacterial pellet from 5 ml of liquid culture was suspended in 0.5 ml of lysis buffer consisting of 0.3 M Tris-HCl, pH 8.0, 0.1 M NaCl, and 6 mM EDTA. The cell suspension was transferred into a conical 2-ml screw-cap vial, which is one-fourth filled with 0.17-mm acid-washed sterile glass beads. Cells were disrupted by vigorous shaking with 0.5 ml of chloroform on a Mini-Bead Beater cell disrupter (Biospec Products, Bartlesville, UK) for 5 min. DNA in the upper layer after centrifugation was further purified by phenol/chloroform extraction, concentrated by ethanol precipitation, and dissolved in 300 ml of TE buffer consisting of 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA. The purified genomic DNA was completely digested with EcoRI and ligated into the EcoRI site of lgt10. Genomic library constructs were introduced into E. coli strain C600hfl by means of in vitro packaging using gigapack plus (Stratagene). Generation of Probe for Screening\u2014The N-terminal amino acid sequence was used to design oligonucleotide primers for amplifying the DNA fragment encoding the N-terminal of azoreductase (Fig. 2A). The reaction mixture (50 ml) consisted of long and accurate (LA) PCR buffer II (Mg21-free); 2.5 mM MgCl2; 200 mM each dATP, dCTP, dGTP, and dTTP; 10 ng of DNA from Bacillus sp. OY2-1; 1.25 units of Takara LA Taq DNA polymerase (Takara Shuzo Co., Ltd., Japan); and 0.5 mM each primer AZR-1 and AZR-2 (Fig. 2A). PCR was carried out for 30 cycles in a Takara Thermal Cycler Personal (Takara Shuzo Co., Ltd.), with each cycle consisting of denaturation for 30 s at 94 \u00b0C, annealing for 30 s at 55 \u00b0C, and extension for 1 min at 72 \u00b0C. The PCR product was extracted from the gel after separation on a 1% agarose gel and was directly subcloned into the pT7Blue(R)T vector. The subclones were sequenced by the dideoxy chain termination method (12) with a Model 310 genetic analyzer (PE Biochemicals Inc). A hybridization probe was synthesized by PCR using a PCR DIG labeling kit (Roche Diagnostics Co., Germany). The reaction mixture (50 ml) consisted of 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 mM each dATP, dCTP, and dGTP and 130 mM dTTP; 70 mM digoxigenin-11-dUTP; 1.25 units of Taq DNA polymerase; a 1 mM concentration of primers M13 M1 & M13 RV (Takara Shuzo Co., Ltd.); and 10 ng of plasmid carrying the PCR product encoding the N-terminal of azoreductase. PCR was performed under the same conditions as described above. Cloning and DNA Sequencing of the Azoreductase Gene\u2014A phage library was screened essentially as previously described (13). Approximately 1 3 105 plaques from the genomic library were plated with E. coli C600hfl and incubated at 37 \u00b0C for 6 h. Nylon filters (Nytran 13N, Schleicher & Schuell Co.) were processed for hybridization. The filters were prehybridized in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.) at 68 \u00b0C for 30 min and then hybridized for 1 h at 48 \u00b0C with a 10 ng/ml concentration of digoxigenin-labeled probe described above in the same buffer as used for prehybridization. The filters were washed for 5 min in 23 SSC and 0.1% SDS at room temperature followed by washing for 15 min in 0.23 SSC and 0.1% SDS at 48 \u00b0C. The hybridized probe was detected after 30 min of incubation at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antibody (Fab; Roche Diagnostics Co.) diluted 1:5000. The enzyme-catalyzed color reaction was carried out using a nitro blue tetrazolium salt (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) system (Wako Pure Chemical Industries, Japan) in Buffer 3 consisting of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2. The DNA inserts in the positive clones were subcloned into the EcoRI site of pUC18 for further characterization. The EcoRI fragment in the subclone was digested by SphI, NlaIV, or HincII; further subcloned in pUC18; and sequenced as shown in Fig. 2 by the dideoxy chain termination method with a Model 310 genetic analyzer. Database Search\u2014Protein and DNA sequences with homology to the deduced amino acid sequence of the azoreductase ORF were searched using TBLASTN from the National Center for Biological Information. Southern Blot Hybridization\u2014Detection of the restriction DNA fragment carrying the azoreductase gene was performed according to Southern (14). One mg of genomic DNA was completely digested with restriction enzymes, separated on a 0.7% agarose gel, and vacuum-transferred to Nytran 13N nylon filters. The filters were prehybridized in ExpressHyb hybridization solution at 68 \u00b0C for 30 min followed by hybridization with the same solution containing a 10 ng/ml Dig-labeled 1.2-kbp EcoRI DNA fragment carrying the whole coding region of azoreductase. After hybridization, the filters were washed for 5 min with 23 SSC and 0.1% SDS at room temperature followed by washing twice with 23 SSC/0.1% SDS for 15 min at 68 \u00b0C. The hybridized Dig-labeled probe on the filters were detected by alkaline phosphatase-conjugated anti-digoxigenin antibody, followed by color development using NBT/BCIP as substrates in Buffer 3. Expression of Azoreductase in E. coli\u2014The entire open reading frame of azoreductase was amplified by PCR. Briefly, the reaction mixture (50 ml) consisted of LA-PCR buffer II (Mg21-free); 2.5 mM MgCl2; 200 mM each dATP, dCTP, dGTP, and dTTP; 10 ng plasmid pT7B-AZR5\u20138; 1.25 units of Takara LA Taq DNA polymerase, and 0.5 mM each of primers AZR-rec-S-Nde (CATATGAAACTAGTCGTTATTAAC) and AZR-rec-E-Xba (TCTAGAGCAGATAGACTATTGGCTCC). PCR was carried out for 30 cycles in a Takara Thermal Cycler Personal, with each cycle consisting of denaturation for 30 s at 94 \u00b0C, annealing for 30 s at 55 \u00b0C, and extension for 1 min at 72 \u00b0C. The PCR product was extracted from the gel after separation on 1% agarose gel electrophoresis and subcloned into pT7Blue(R)T for confirmation of the nucleotide sequence and then transferred into expression vectors pTrx-Fus or pTrcNd after digestion by NdeI and XbaI. Expression of reductase in pTrx-Fus system was performed by adding tryptophan at a concentration of 0.1 mg/ml in ID medium. Expression in pTrcNd was performed by adding isopropyl-b-D-thio-galactopyranoside (IPTG) at a concentration of 1 mM in Luria-Bertani medium. Cells from 10 ml of induced culture were suspended in 0.5 ml of 20 mM sodium phosphate buffer, pH 7.0, lysed by two cycles of freezing at 280 \u00b0C and thawed at 37 \u00b0C followed by sonication (15 s, 70% output, 103). The supernatants from a 9000 3 g, 30 min centrifugation were used directly for enzyme assay or SDS-PAGE analysis. Purification of Recombinant Azoreductase by Red-Sepharose CL-6B\u2014The cells from 200 ml of culture were suspended in 20 ml of 20 mM sodium-phosphate buffer, pH 7.0 and lysed by freezing and thawing followed by sonication (15 s, 70% output, 103). After centrifugation at 9000 3 g for 30 min, the supernatant was applied to a Red-Sepharose CL-6B column (Amersham Pharmacia Biotech) followed by washing with 20 mM sodium phosphate buffer, pH 7.0. The recombinant azoreductase was eluted from the column with 10 mM b-NADH. The eluate was dialyzed against two changes of 1000 volumes of 20 mM sodium phosphate buffer, pH 7.0 and used for enzyme assay. Enzyme Assays\u2014Azo dye-reducing activity was analyzed by measuring the decrease in optical density at suitable wavelengths with a Hitachi U 3300 spectrophotometer at various temperatures basically according to Pasti-Grigsby et al. (15). The reaction mixture in a total volume of 1.0 ml consisted of various concentrations of azo dyes (Roccelin, Solar Orange, Sumifix Black B; shown in Fig. 3) in 20 mM sodium phosphate buffer, pH 7.0, and bacterial lysates or purified enzyme. The reaction mixture was preincubated for 5 min at the assay temperature, and the reaction was started by the addition of 25 ml of various concentrations of b-NADPH. The enzymatic activities were measured by the decrease in optical density at optimal wavelengths. The enzyme activity was expressed as the amount of reduced dye per min with 1 mg of enzyme. Kinetic parameters for the reduction of each dye by native and recombinant azoreductases were estimated by nonlinear regression analysis according to Shimada et al. (16)."
  },
  "38740275": {
    "bleu": 92.70795163063393,
    "meteor": 94.38069344148789,
    "rouge1_p": 0.9644690090801421,
    "rouge1_r": 0.9760287654814223,
    "rouge1_f": 0.9702144559173946,
    "rouge2_p": 0.9447077409162717,
    "rouge2_r": 0.95603517186251,
    "rouge2_f": 0.9503377036154151,
    "rougeL_p": 0.959731543624161,
    "rougeL_r": 0.9712345185777067,
    "rougeL_f": 0.9654487688641777,
    "cosine_similarity": 0.9297889975813739,
    "precision": 0.8130841121495327,
    "recall": 0.87,
    "ner_f1": 0.8405797101449274,
    "ner_tp": 174,
    "ner_fp": 40,
    "ner_fn": 26,
    "bertscore_p": 0.958568811416626,
    "bertscore_r": 0.9630984663963318,
    "bertscore_f1": 0.9609882831573486,
    "bertscore_scibert_error": "The expanded size of the tensor (3557) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 3557].  Tensor sizes: [1, 512]",
    "len_gt": 14286,
    "len_jl": 14460,
    "GT": "Experimental procedures General products and instruments. Enzyme kinetics and UV-Visible absorption profiles were determined using a Cary 3500 spectrophotometer (Agilent Technologies). Cell suspensions and lysate were centrifuged using a 5910 Ri (Eppendorf) or a Microcentrifuge 24 (USA Scientific). Protein Expression. A codon-optimized gene of UniProt A0A1M6LNR3_9FIRM from Anaerotignum lactatifermentan DSM 14214, AlAzoR, cloned into pET28b was purchased from Twist Bioscience (the gene sequence can be found in Table S3). The plasmid was transformed into competent autolysis E. coli strain XJb (DE3) according to the manufacturer\u2019s protocol (Zymo Research). A pET15b plasmid containing the thermostable phosphite dehydrogenase gene, 17x-PTDH, was obtained from Addgene (plasmid #166786). Liquid cultures of transformed cells were prepared and grown overnight at 37\u00b0C in 5 mL LB culture with the appropriate antibiotics. To overexpress the protein, 500 microliters of the overnight culture were added to 50 mL sterile ZYM-5052 media (or 10 mL of overnight culture into 1000 mL media) supplemented with 0.3 mM arabinose and 60 \u03bcg/mL kanamycin when expressing AlAzoR and 100 \u03bcg/mL ampicillin for PTDH.(51) Cultures were grown at 37\u00b0C overnight and centrifuged at 4,000 \u00d7 g at 4\u00b0C for 15 minutes. Cells were resuspended in 5 milliliters of Ni-NTA wash buffer (0.5 M NaCl, 50 mM sodium phosphate, 50 mM imidazole, pH 8.0) per mass of cells and stored at \u221220\u00b0C until purified. Protein Purification. Following autoinduction frozen cells (approximately 16.5 g from 2 liters of media, resuspended in 82.5 mL Ni-NTA wash buffer) were thawed at 37\u00b0C. When fully thawed, lysozyme solution was added to 0.5 mg/mL, and underwent a second freeze-thaw cycle from \u221280\u00b0C to 37\u00b0C. Cells were then spun down at 15,000 \u00d7 g and 4\u00b0C for 45 minutes, and the clarified supernatant was added to 15 mL of HisPur\u2122 Ni-NTA Resin (Thermo Fisher Scientific) equilibrated with Ni-NTA wash buffer in 50 mL tubes. The resin and supernatant were placed on an end-over-end rotator for 15 minutes at 4 \u00b0C and spun down at 500 \u00d7 g for 2 minutes. The supernatant was removed, 40 mL Ni-NTA wash buffer was added and the resin and wash buffer were put back on the end-over-end rotator for 15 minutes at 4\u00b0C. The resin and wash buffer were then transferred to a disposable column. The column was then repeatedly washed with Ni-NTA wash buffer until no trace protein was detected in the flow through by a Bradford Assay (Bio-Rad). Bound protein was eluted using Ni-NTA elution buffer (500 mM NaCl, 50 mM sodium phosphate, 250 mM imidazole, pH 8.0). Purification was verified by SDS-PAGE using a Mini-PROTEAN TGX gel (Bio-Rad) (Figure S8). Collected protein samples were dialyzed overnight at 4\u00b0C in 50 mM sodium phosphate buffer at pH 7.2. Cofactor Determination. The specific flavin cofactor for AlAzoR was determined by taking a spectral scan of a 3 mL-sample of AlAzoR in 25 mM ammonium bicarbonate buffer at pH 7.8 (A280 = 1.391). The protein was denatured through heating from 20\u00b0C to 90\u00b0C with a ramp rate of 5 \u00b0C per minute. Another spectral scan was taken of the now denatured sample, and the extinction coefficients are listed in Table 4 to determine the concentration and flavin cofactor was present. A sample of this heated protein was spun in a 1.5 mL microcentrifuge tube at 15,000 \u00d7 g for 10 minutes to pellet the protein. The supernatant was then spun through a Pierce Concentrator polyethersulfone columns (10K molecular weight cut-off, Thermo Scientific) for 10 minutes at 15,000 \u00d7 g, and the flowthrough was submitted for mass spectrometry analysis. Enzyme Kinetics. The activity of AlAzoR with its various substrates was measured through a kinetic assay that was conducted in 3 mL of 50 mM MOPS buffer at pH 7.2 over the course of one minute at 22\u00b0C. The assay contained a constant amount of either 40 or 400 \u03bcM NADH and 3.2\u201320 \u03bcL of substrate solutions prepared in DMSO. Concentrated substrate stock solutions were prepared and diluted 10-fold, to extend the tested substrate range while minimizing error. Upon addition of AlAzoR (in 50 mM sodium phosphate pH 7.2 buffer), the change in absorbance of substrate over time was measured in triplicate at each substrate concentration. The rates were converted to the change in concentration per second per concentration of the enzyme, and fitted to a nonlinear curve using Wolfram Mathematica (Mathematica, Version 13.2). This curve allowed for kinetic constants to be estimated using the modified Michaelis-Menten kinetic equation(52): v= \ud835\udc58c\u2062a\u2062t\u2061/\u2062\ud835\udc3eM\u2061[S] 1+(\ud835\udc58c\u2062a\u2062t\u2061/\u2062\ud835\udc3eM\u2061[S])/\ud835\udc58c\u2062a\u2062t = \ud835\udc58S\u2062P\u2061[S] 1+(\ud835\udc58S\u2062P\u2061[S])/\ud835\udc58c\u2062a\u2062t Determining the Extinction Coefficient of Enzyme. The concentration of AlAzoR was determined using the absorbance at 445 of the boiled sample of AlAzoR with the \ud835\udf00445 of FMN to determine the concentration of FMN. The concentration of FMN was then converted to the expected absorbance of FMN at 280 nm using the \ud835\udf00280 of FMN. The calculated FMN absorbance at 280 nm was then subtracted from the absorbance measured of the boiled sample of AlAzoR at 280 nm to calculate absorbance at 280 nm due to the protein. Using the theoretical protein \ud835\udf00280 of 24410 M\u22121\u2062c\u2062m\u22121 determined by the ProtParam webtool (https://web.expasy.org/protparam/), the concentration of the protein in the boiled AlAzoR sample was determined. The relative concentration of FMN and protein can determine the percentage of holoprotein that could be active in the reduction reaction. This was then converted into an extinction coefficient for the AlAzoR using the equation below involving the \ud835\udf00280 of FMN (16,200\u2062M\u22121\u2062c\u2062m\u22121), the theoretical \ud835\udf00280 of AlAzoR, and the percent of protein loaded with FMN. A280=Cprotein\u2062(16200\u2062M\u22121\u2062c\u2062m\u22121(\u2062h\u2061o\u2062l\u2062o\u2062%)+24410\u2062M\u22121\u2062c\u2062m\u22121) AlAzoR kinetics was measured using a batch of enzyme that was 53.7% holo protein, resulting in an effective extinction coefficient of 31,000 M\u22121cm\u22121. This new extinction coefficient can be used with a DS-11FX+ Spectrophotometer (DeNovix) to determine the concentration of the AlAzoR that is loaded with flavin in a given sample. The concentration of AlAzoR was kept low to keep visible light absorbance between 400\u2013500 nm below 0.02 AU. Ethyl Red. The activity of AlAzoR with ethyl red was measured in an assay containing 40 or 400 \u03bcM NADH and varied concentrations of ethyl red, ranging from 4.8 \u03bcM to 180 \u03bcM by using either a 4.5 mM or 45 mM ethyl red stock in DMSO. After the addition of 0.552 \u03bcM AlAzoR, the change in absorbance at 453 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (23,300 M\u22121cm\u22121). Methyl Red. The activity of AlAzoR with methyl red was measured in an assay containing 400 \u03bcM NADH and varied concentrations of methyl red, ranging from 2.4 \u03bcM to 150 \u03bcM by using either a 2.25 mM or 22.5 mM methyl red stock in DMSO. After the addition of 0.400 \u03bcM AlAzoR, the change in absorbance at 438 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (17,300 M\u22121cm\u22121). Indigo Carmine. The activity of AlAzoR with indigo carmine was measured in an assay containing 400 \u03bcM NADH and varied concentrations of indigo carmine, ranging from 24.2 \u03bcM-241.9 \u03bcM by using either a 3.75 mM or 37.5 mM indigo carmine stock in DMSO. After the addition of 0.243 \u03bcM AlAzoR, the change in absorbance at 612 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (11,400 M\u22121cm\u22121). Sulfasalazine. The activity of AlAzoR with sulfasalazine was measured in an assay containing 40 \u03bcM NADH and varied concentrations of sulfasalazine, ranging from 3.04 \u03bcM to 190 \u03bcM by using either a 2.85 mM or 28.5 mM sulfasalazine stock in DMSO. After the addition of 0.243 \u03bcM AlAzoR, the change in absorbance at 383 nm (off \u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (15,800 M\u22121cm\u22121). Phenazopyridine: The activity of AlAzoR with phenazopyridine was measured in an assay containing 400 \u03bcM NADH and varied concentrations of phenazopyridine, ranging from 1.92 \u03bcM to 120 \u03bcM by using either a 1.8 mM or 18 mM phenazopyridine stock in DMSO. After the addition of 0.644 \u03bcM AlAzoR, the change in absorbance at 428 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (22,300 M\u22121cm\u22121). NADH: The activity of AlAzoR with NADH and NADPH was measured in an assay containing 120 \u03bcM ethyl red and varied concentrations of NADH or NADPH, ranging from 6.4 \u03bcM to 400 \u03bcM by using either a 6 mM or 60 mM stock in 50 mM MOPS buffer (pH 7.2). After the addition of 0.400 \u03bcM AlAzoR, the change in absorbance at 453 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (23,300 M\u22121cm\u22121). Phenol blue: The activity of AlAzoR with phenol blue was measured in an assay containing 400 \u03bcM NADH and varied concentrations of phenol blue by using a 1.875 mM or 18.75 mM stock in DMSO. After the addition of 0.644 \u03bcM AlAzoR, the change in absorbance at 340 nm (due to changes in NADH concentration) was measured over time and converted to changes in concentration using the extinction coefficient 6,220 M\u22121cm\u22121. These rates were corrected for the background rate of absorbance change at 340 nm in the presence of NADH but in the absence of phenol blue. 5-hydroxy-2-methylnaphthalene-1,4-dione (HMND): The specific activity of AlAzoR with HMND was measured in an assay containing 400 \u03bcM NADH and varied concentrations of HMND by using a 1.875 mM or 18.75 mM stock in DMSO. After the addition of 0.644 \u03bcM AlAzoR, the change in absorbance at 340 nm (due to changes in NADH concentration) was measured over time and converted to changes in concentration using the extinction coefficient 6,220 M\u22121cm\u22121. These rates were corrected for the background rate of absorbance change at 340 nm in the presence of NADH but in the absence of HMND. NMR Spectroscopy. For sulfasalazine, phenazopyridine, and methyl red, 1.0 mL reaction mixtures were made containing 0.1 mM NADH, 10 mM azo-substrate, 50 mM phosphite, 100 nM AlAzoR (from a 69.03 \u03bcM stock in sodium phosphate buffer), 3.5 \u03bcM phosphite dehydrogenase (from a 8.84 \u03bcM 17x-PTDH stock in sodium phosphate buffer) in 5% d6-DMSO in 50 mM sodium phosphate buffer (pH 7.2). The reaction mixtures were incubated overnight and analyzed by 1H NMR (Bruker Ascend 400 MHz) with solvent suppression at the water peak. Due to difficulties with solubility, the indigo carmine reaction was conducted using the following assay conditions: 0.21 \u03bcM NADH, 0.74 \u03bcM of phosphite dehydrogenase (from a 8.84 \u03bcM 17x-PTDH stock in sodium phosphate buffer), and 5.74 \u03bcM of AlAzoR (from a 69.03 \u03bcM stock in sodium phosphate buffer) in 4 mL of 50 mM sodium phosphate buffer (pH 7.2). The reaction ran overnight at room temperature in a covered beaker. An extraction was performed with 3 increments of 2 mL of ethyl acetate in a separatory funnel, using sodium sulfate as a drying agent. Solvent was removed, and the residue was dissolved in 750 \u03bcL of d6-DMSO. Mass Spectrometry. AlAzoR activity against 27 different substrates was tested through LC-ESI-MS (Table S1). Each reaction contained 0.721 \u03bcM AlAzoR, 400 mM NADH, and 182 \u03bcM substrate in 50 mM Ammonium bicarbonate buffer (pH 7.8) After an overnight incubation at ambient temperature, the samples were spun down in Pierce Concentrator PES columns (10K MWCO) (Thermo Scientific) for 5 minutes at 15,000 \u00d7 g. The flow through of the column was then transferred into autosampler vials to be analyzed by LC-ESI mass spectrometry (Brown University). Resulting ions are reported in Table S2. Sequence Similarity Network and Chemically-Guided Functional Profiling. The EFI-EST web tool (https://efi.igb.illinois.edu/efi-est/) was used to generate an SSN using the \u201cFamiles\u201d (Option B) tab, using the Flavodoxin_fold protein family IPR003680 and the sequences released in the UniProt: 2023\u201305 / InterPro: 97.0 dataset. Fragments were filtered using a minimum length cutoff of 165 amino acids. The UniRef90 cluster IDs (containing UniProt IDs that are 90% identical or more to a reference sequence) were used to convert the 63,672 proteins into 27,716 metanodes (representing one sequence or more). These nodes were segregated into isofunctional clusters using an alignment score threshold of 80 (corresponding to ~65% amino acid identity) following the substrate preferences identified in Suzuki, the references therein, and others found in the literature.(17, 18, 28\u201330, 32\u201342) This alignment score is similar in value to the negative base ten logarithm of the \u201ce-value\u201d obtained by basic local alignments, but provides a database size-independent value. This SSN was then sent to the EFI-CGFP web tool (https://efi.igb.illinois.edu/efi-cgfp/) for chemically-guided functional profiling (CGFP) using the default methods of the tool.(27, 28) This tool creates markers using the ShortBRED algorithm.(53) Briefly, proteins that are at least 85% identical are aligned, a consensus sequence is created, and markers are created from these sequences after ensuring they are unique. These markers are compared to the NIH HMP-1 dataset and are used to calculate the metagenome abundance. The abundance of the protein-coding genes is reported as the median abundance if multiple markers are identified, and metagenome abundances are normalized by average genome size.(54) The proteins identified in the HMP-1 metagenomes are at least 85% identical to the proteins identified in the SSN provided in Figure 5a. Computational Modeling. In PyMOL, an AlphaFold model of AlAzoR (AF-A0A1M6LNR3-F1) was aligned to the Pseudomonas aeruginosa azoreductase PaAzoR1 bound to methyl red (PDB: 2V9C) to create a dimeric model of AlAzoR bound to FMN and methyl red. Potential substrates were docked in the methyl red binding site of the PaAzoR1 structure and the predicted AlAzoR model using Autodock on Nanome.(55) In each case, the crystallographic methyl red was removed, and substrates were docked into the methyl red binding site.",
    "LLM": "3.1. Experimental procedures General products and instruments. Enzyme kinetics and UV\u2013Visible absorption profiles were determined using a Cary 3500 spectrophotometer (Agilent Technologies). Cell suspensions and lysate were centrifuged using a 5910 Ri (Eppendorf) or a Microcentrifuge 24 (USA Scientific). Protein Expression. A codon-optimized gene of UniProt A0A1M6LNR3_9FIRM from Anaerotignum lactatifermentan DSM 14214, AlAzoR, cloned into pET28b was purchased from Twist Bioscience (the gene sequence can be found in Table S3). The plasmid was transformed into competent autolysis E. coli strain XJb (DE3) according to the manufacturer\u2019s protocol (Zymo Research). A pET15b plasmid containing the thermostable phosphite dehydrogenase gene, 17x-PTDH, was obtained from Addgene (plasmid #166786). Liquid cultures of transformed cells were prepared and grown overnight at 37 \u25e6C in 5 mL LB culture with the appropriate antibiotics. To overexpress the protein, 500 \u03bcL of the overnight culture were added to 50 mL sterile ZYM-5052 media (or 10 mL of overnight culture into 1000 mL media) supplemented with 0.3 mM arabinose and 60 \u03bcg/mL kanamycin when expressing AlAzoR and 100 \u03bcg/mL ampicillin for PTDH [51]. Cultures were grown at 37 \u25e6C overnight and centrifuged at 4000\u00d7g at 4 \u25e6C for 15 min. Cells were resuspended in 5 mL of Ni-NTA wash buffer (0.5 M NaCl, 50 mM sodium phosphate, 50 mM imidazole, pH 8.0) per mass of cells and stored at \u2212 20 \u25e6C until purified. Protein Purification. Following autoinduction frozen cells (approximately 16.5 g from 2 L of media, resuspended in 82.5 mL Ni-NTA wash buffer) were thawed at 37 \u25e6C. When fully thawed, lysozyme solution was added to 0.5 mg/mL, and underwent a second freeze-thaw cycle from \u2212 80 \u25e6C to 37 \u25e6C. Cells were then spun down at 15,000\u00d7g and 4 \u25e6C for 45 min, and the clarified supernatant was added to 15 mL of HisPur\u2122 Ni-NTA Resin (Thermo Fisher Scientific) equilibrated with Ni-NTA wash buffer in 50 mL tubes. The resin and supernatant were placed on an end-over-end rotator for 15 min at 4 \u25e6C and spun down at 500\u00d7g for 2 min. The supernatant was removed, 40 mL Ni-NTA wash buffer was added and the resin and wash buffer were put back on the end-over-end rotator for 15 min at 4 \u25e6C. The resin and wash buffer were then transferred to a disposable column. The column was then repeatedly washed with Ni-NTA wash buffer until no trace protein was detected in the flow through by a Bradford Assay (Bio-Rad). Bound protein was eluted using Ni-NTA elution buffer (500 mM NaCl, 50 mM sodium phosphate, 250 mM imidazole, pH 8.0). Purification was verified by SDS-PAGE using a Mini-PROTEAN TGX gel (Bio-Rad) (Fig. S8). Collected protein samples were dialyzed overnight at 4 \u25e6C in 50 mM sodium phosphate buffer at pH 7.2. Cofactor Determination. The specific flavin cofactor for AlAzoR was determined by taking a spectral scan of a 3 mL-sample of AlAzoR in 25 mM ammonium bicarbonate buffer at pH 7.8 (A280 = 1.391). The protein was denatured through heating from 20 \u25e6C to 90 \u25e6C with a ramp rate of 5 \u25e6C per minute. Another spectral scan was taken of the now denatured sample, and the extinction coefficients are listed in Table 5 to determine the concentration and flavin cofactor was present. A sample of this heated protein was spun in a 1.5 mL microcentrifuge tube at 15,000\u00d7g for 10 min to pellet the protein. The supernatant was then spun through a Pierce Concentrator polyethersulfone columns (10K molecular weight cut-off, Thermo Scientific) for 10 min at 15,000\u00d7g, and the flowthrough was submitted for mass spectrometry analysis. Enzyme Kinetics. The activity of AlAzoR with its various substrates was measured through a kinetic assay that was conducted in 3 mL of 50 mM MOPS buffer at pH 7.2 over the course of 1 min at 22 \u25e6C. The assay contained a constant amount of either 40 or 400 \u03bcM NADH and 3.2\u201320 \u03bcL of substrate solutions prepared in DMSO. Concentrated substrate stock solutions were prepared and diluted 10-fold, to extend the tested substrate range while minimizing error. Upon addition of AlAzoR (in 50 mM sodium phosphate pH 7.2 buffer), the change in absorbance of substrate over time was measured in triplicate at each substrate concentration. The rates were converted to the change in concentration per second per concentration of the enzyme, and fitted to a nonlinear curve using Wolfram Mathematica (Mathematica, Version 13.2). This curve allowed for kinetic constants to be estimated using the modified Michaelis-Menten kinetic equation where kSP = kcat/KM [52]: v = kcat/KM [S] 1 + ( kcat/KM [S] )/kcat = kSP[S] 1 + (kSP[S])/kcat Determining the Extinction Coefficient of Enzyme. The concentration of AlAzoR was determined using the absorbance at 445 nm of the boiled sample of AlAzoR with the \u03b5445 of FMN to determine the concentration of FMN. The concentration of FMN was then converted to the expected absorbance of FMN at 280 nm using the \u03b5280 of FMN. The calculated FMN absorbance at 280 nm was then subtracted from the absorbance measured of the boiled sample of AlAzoR at 280 nm to calculate absorbance at 280 nm due to the protein. Using the theoretical protein \u03b5280 of 24,410 M\u2212 1 cm\u2212 1 determined by the ProtParam webtool (https://web.expasy.org/protparam/), the concentration of the protein in the boiled AlAzoR sample was determined. The relative concentration of FMN and protein can determine the percentage of holoprotein that could be active in the reduction reaction. This was then converted into an extinction coefficient for the AlAzoR using the equation below involving the \u03b5280 of FMN (40,610 M\u2212 1 cm\u2212 1), the theoretical \u03b5280 of AlAzoR, and the percent of protein loaded with FMN (holo%). A280 = Cprotein( 16100M\u2212 1cm\u2212 1( holo%) + 24410M\u2212 1cm\u2212 1) AlAzoR kinetics was measured using a batch of enzyme that was 53.7 % holo protein, resulting in an effective extinction coefficient of 31,000 M\u2212 1 cm\u2212 1. This new extinction coefficient can be used with a DS-11FX + Spectrophotometer (DeNovix) to determine the concentration of the AlAzoR that is loaded with flavin in a given sample. The concentration of AlAzoR was kept low to keep visible light absorbance between 400 and 500 nm below 0.02 AU. Ethyl Red. The activity of AlAzoR with ethyl red was measured in an assay containing 40 or 400 \u03bcM NADH and varied concentrations of ethyl red, ranging from 4.8 \u03bcM to 180 \u03bcM by using either a 4.5 mM or 45 mM ethyl red stock in DMSO. After the addition of 0.552 \u03bcM AlAzoR, the change in absorbance at 453 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (23,300 M\u2212 1 cm\u2212 1). Methyl Red. The activity of AlAzoR with methyl red was measured in an assay containing 400 \u03bcM NADH and varied concentrations of methyl red, ranging from 2.4 \u03bcM to 150 \u03bcM by using either a 2.25 mM or 22.5 mM methyl red stock in DMSO. After the addition of 0.400 \u03bcM AlAzoR, the change in absorbance at 438 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (17,300 M\u2212 1 cm\u2212 1). Indigo Carmine. The activity of AlAzoR with indigo carmine was measured in an assay containing 400 \u03bcM NADH and varied concentrations of indigo carmine, ranging from 24.2 \u03bcM to 241.9 \u03bcM by using either a 3.75 mM or 37.5 mM indigo carmine stock in DMSO. After the addition of 0.243 \u03bcM AlAzoR, the change in absorbance at 612 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (11,400 M\u2212 1 cm\u2212 1). Sulfasalazine. The activity of AlAzoR with sulfasalazine was measured in an assay containing 40 \u03bcM NADH and varied concentrations of sulfasalazine, ranging from 3.04 \u03bcM to 190 \u03bcM by using either a 2.85 mM or 28.5 mM sulfasalazine stock in DMSO. After the addition of 0.243 \u03bcM AlAzoR, the change in absorbance at 383 nm (off \u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (15,800 M\u2212 1 cm\u2212 1). Phenazopyridine: The activity of AlAzoR with phenazopyridine was measured in an assay containing 400 \u03bcM NADH and varied concentrations of phenazopyridine, ranging from 1.92 \u03bcM to 120 \u03bcM by using either a 1.8 mM or 18 mM phenazopyridine stock in DMSO. After the addition of 0.644 \u03bcM AlAzoR, the change in absorbance at 428 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (22,300 M\u2212 1 cm\u2212 1). NADH: The activity of AlAzoR with NADH and NADPH was measured in an assay containing 120 \u03bcM ethyl red and varied concentrations of NADH or NADPH, ranging from 6.4 \u03bcM to 400 \u03bcM by using either a 6 mM or 60 mM stock in 50 mM MOPS buffer (pH 7.2). After the addition of 0.400 \u03bcM AlAzoR, the change in absorbance at 453 nm (\u03bb-max) was measured over time and converted to changes in concentration using the experimentally determined extinction coefficient (23,300 M\u2212 1 cm\u2212 1). Phenol blue: The activity of AlAzoR with phenol blue was measured in an assay containing 400 \u03bcM NADH and varied concentrations of phenol blue by using a 1.875 mM or 18.75 mM stock in DMSO. After the addition of 0.644 \u03bcM AlAzoR, the change in absorbance at 340 nm (due to changes in NADH concentration) was measured over time and converted to changes in concentration using the extinction coefficient 6220 M\u2212 1 cm\u2212 1. These rates were corrected for the background rate of absorbance change at 340 nm in the presence of NADH but in the absence of phenol blue. 5-hydroxy-2-methylnaphthalene-1,4-dione (HMND): The specific activity of AlAzoR with HMND was measured in an assay containing 400 \u03bcM NADH and varied concentrations of HMND by using a 1.875 mM or 18.75 mM stock in DMSO. After the addition of 0.644 \u03bcM AlAzoR, the change in absorbance at 340 nm (due to changes in NADH concentration) was measured over time and converted to changes in concentration using the extinction coefficient 6220 M\u2212 1 cm\u2212 1. These rates were corrected for the background rate of absorbance change at 340 nm in the presence of NADH but in the absence of HMND. NMR Spectroscopy. For sulfasalazine, phenazopyridine, and methyl red, 1.0 mL reaction mixtures were made containing 0.1 mM NADH, 10 mM azo-substrate, 50 mM phosphite, 100 nM AlAzoR (from a 69.03 \u03bcM stock in sodium phosphate buffer), 3.5 \u03bcM phosphite dehydrogenase (from a 8.84 \u03bcM 17x-PTDH stock in sodium phosphate buffer) in 5 % DMSO\u2011d6 in 50 mM sodium phosphate buffer (pH 7.2). The reaction mixtures were incubated overnight and analyzed by 1H NMR (Bruker Ascend 400 MHz) with solvent suppression at the water peak. Due to difficulties with solubility, the indigo carmine reaction was conducted using the following assay conditions: 0.21 \u03bcM NADH, 0.74 \u03bcM of phosphite dehydrogenase (from a 8.84 \u03bcM 17x-PTDH stock in sodium phosphate buffer), and 5.74 \u03bcM of AlAzoR (from a 69.03 \u03bcM stock in sodium phosphate buffer) in 4 mL of 50 mM sodium phosphate buffer (pH 7.2). The reaction ran overnight at room temperature in a covered beaker. An extraction was performed with 3 increments of 2 mL of ethyl acetate in a separatory funnel, using sodium sulfate as a drying agent. Solvent was removed, and the residue was dissolved in 750 \u03bcL of DMSO\u2011d6. Mass Spectrometry. AlAzoR activity against 27 different substrates was tested through LC-ESI-MS (Table S1). Each reaction contained 0.721 \u03bcM AlAzoR, 400 mM NADH, and 182 \u03bcM substrate in 50 mM Ammonium bicarbonate buffer (pH 7.8) After an overnight incubation at ambient temperature, the samples were spun down in Pierce Concentrator PES columns (10K MWCO) (Thermo Scientific) for 5 min at 15,000\u00d7g. The flow through of the column was then transferred into autosampler vials to be analyzed by LC-ESI mass spectrometry (Brown University). Resulting ions are reported in Table S2. Sequence Similarity Network and Chemically-Guided Functional Profiling. The EFI-EST web tool (https://efi.igb.illinois.edu/efi-est/) was used to generate an SSN using the \u201cFamiles\u201d (Option B) tab, using the Flavodoxin_fold protein family IPR003680 and the sequences released in the UniProt: 2023\u201305/InterPro: 97.0 dataset. Fragments were filtered using a minimum length cutoff of 165 amino acids. The UniRef90 cluster IDs (containing UniProt IDs that are 90% identical or more to a reference sequence) were used to convert the 63,672 proteins into 27,716 metanodes (representing one sequence or more). These nodes were segregated into isofunctional clusters using an alignment score threshold of 80 (corresponding to ~65 % amino acid identity) following the substrate preferences identified in Suzuki, the references therein, and others found in the literature [17,18,28\u201330, 32\u201342]. This alignment score is similar in value to the negative base ten logarithm of the \u201ce-value\u201d obtained by basic local alignments, but provides a database size-independent value. This SSN was then sent to the EFI-CGFP web tool (https://efi.igb.illinois.edu/efi-cgfp/) for chemically-guided functional profiling (CGFP) using the default methods of the tool [27,28]. This tool creates markers using the ShortBRED algorithm [53]. Briefly, proteins that are at least 85% identical are aligned, a consensus sequence is created, and markers are created from these sequences after ensuring they are unique. These markers are compared to the NIH HMP-1 dataset and are used to calculate the metagenome abundance. The abundance of the protein-coding genes is reported as the median abundance if multiple markers are identified, and metagenome abundances are normalized by average genome size [54]. The proteins identified in the HMP-1 metagenomes are at least 85 % identical to the proteins identified in the SSN provided in Fig. 5a. Computational Modeling. In PyMOL, an AlphaFold model of AlAzoR (AF-A0A1M6LNR3-F1) was aligned to the Pseudomonas aeruginosa azoreductase PaAzoR1 bound to methyl red (PDB: 2V9C) to create a dimeric model of AlAzoR bound to FMN and methyl red. Potential substrates were docked in the methyl red binding site of the PaAzoR1 structure and the predicted AlAzoR model using Autodock on Nanome [55]. In each case, the crystallographic methyl red was removed, and substrates were docked into the methyl red binding site. Table 5 Flavin cofactor extinction coefficients. Wavelength (nm) FMN \u03b5 (M\u2212 1 cm\u2212 1 ) FAD \u03b5 (M\u2212 1 cm\u2212 1 ) 280 16,200 16,000 375 9000 7200 445 10,700 8900 450 10,600 9000"
  },
  "15003265": {
    "bleu": 95.46705046844643,
    "meteor": 95.00438654125757,
    "rouge1_p": 0.9588059701492537,
    "rouge1_r": 0.9792682926829268,
    "rouge1_f": 0.9689291101055807,
    "rouge2_p": 0.929510155316607,
    "rouge2_r": 0.949359365466748,
    "rouge2_f": 0.9393299124660428,
    "rougeL_p": 0.9576119402985075,
    "rougeL_r": 0.9780487804878049,
    "rougeL_f": 0.9677224736048265,
    "cosine_similarity": 0.9346185777742735,
    "precision": 0.875,
    "recall": 0.8926174496644296,
    "ner_f1": 0.883720930232558,
    "ner_tp": 133,
    "ner_fp": 19,
    "ner_fn": 16,
    "bertscore_p": 0.9616283178329468,
    "bertscore_r": 0.969954252243042,
    "bertscore_f1": 0.9659197926521301,
    "bertscore_scibert_error": "The expanded size of the tensor (2294) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2294].  Tensor sizes: [1, 512]",
    "len_gt": 9389,
    "len_jl": 9391,
    "GT": "Materials and methods Bacterial strains, plasmids, and growth conditions Enterococcus faecalis ATCC 19433 was grown in brain heart infusion (BHI) broth or on BHI agar plates and used for inoculum and genomic DNA preparation. E. coli TOP10F\u2032 (Invitrogen), NovaBlue (DE3) (Novagen), and BL21-Gold(DE3)pLysS (Stratagene) were used for recombinant DNA studies. E. coli strains were cultured at 37 \u00b0C in Luria\u2013Bertani (LB) medium with appropriate antibiotics (50 \u00b5g/ml). The plasmids pCR2.1-TOPO (Invitrogen) and pET-11a (Stratagene) were used for cloning and expression, respectively. Culture conditions for E. faecalis to grow in various azo dyes Enterococcus faecalis from a BHI agar plate was inoculated into 20 ml BHI medium supplemented with individual azo dye at a final concentration of 0.2 mM and incubated at 37 \u00b0C for 20 h without shaking in air. The cells were harvested by centrifugation at 5000g. The supernatants were used to assay the azo dye reduction by measuring residual absorption at the appropriate wavelength for each azo dye, as described in the enzyme assay description below. The cells were suspended in 1 ml of 25 mM sodium phosphate buffer, pH 7.1, and disrupted mechanically with sea-sand using a Vortex Gene2 (Scientific Industries) for 15 min at 4 \u00b0C. The mixtures were then centrifuged at 14,000g for 10 min at 4 \u00b0C and the supernatant fraction was collected and used as cell-free extract. Cloning of E. faecalis azoA gene and expression of AzoA in E. coli Genomic DNA of E. faecalis was isolated essentially as described by Wang et al. [26]. Plasmids from E. coli Top10F\u2032 and NovaBlue (DE3) were isolated using a Qiaprep Spin Miniprep kit (Qiagen). A DNA fragment containing the putative E. faecalis azoA was obtained by PCR with the genomic DNA of E. faecalis used as template. The forward primer included a NdeI site before the start codon: 5\u2032-gattcatatgtcaaaattattagttg-3\u2032 (Efs-forward). The reverse primer included a BamHI site downstream of azoA: 5\u2032-cgggatccgctttatgccgcagctaag-3\u2032 (Efs-reverse). PCR was performed in a Mastercycler gradient (Eppendorf). The amplification conditions were one cycle of 95 \u00b0C for 3 min, 30 cycles with each cycle including 30 s of melting at 95 \u00b0C, 30 s of annealing at 50 \u00b0C, and 60 s of extension at 72 \u00b0C, and one final extension cycle at 72 \u00b0C for 5 min. The PCR products were examined by 1% agarose gel electrophoresis. The PCR products amplified from the genomic DNA were directly cloned into pCR2.1-TOPO vector and sequenced. For over-expression of native AzoA in E. coli, the amplified PCR products were cleaved with NdeI and BamHI (New England BioLabs). The digested DNA was purified from the agarose gel and ligated into pET-11a with a rapid DNA ligation kit (Roche). E. coli NovaBlue (DE3) was transformed with the resulting plasmids. The plasmids (pAZOA) were subsequently isolated and introduced into E. coli BL21-Gold(DE3)pLysS by transformation. DNA sequence analysis, translation, and alignment with related genes and proteins were carried out using the Lasergene program (Version 5, DNASTAR). The GenBank program BLAST was utilized to find similar genes or proteins. Enzyme assays Azoreductase activity was assayed by measuring the decrease in optical density at suitable wavelengths with a Hewlett\u2013Packard 8453 UV\u2013visible spectrophotometer at room temperature (23 \u00b0C). A typical reaction mixture (2.0 ml) contained 25 mM potassium phosphate buffer (pH 7.1), 25 \u00b5M azo dye, 0.1 mM NADH, 10 \u00b5M FMN, and a suitable amount of enzyme. The reaction was initiated by addition of the enzyme. Initial velocity was determined by monitoring the change in the amount of substrate in the first 2 min in a glass cuvette of 1.0-cm light path. The following molar absorption coefficients were used: 23,360 M\u207b\u00b9 cm\u207b\u00b9 (Methyl red at 430 nm), 18,200 M\u207b\u00b9 cm\u207b\u00b9 (Orange II at 482 nm), 22,600 M\u207b\u00b9 cm\u207b\u00b9 (Amaranth at 520 nm), 20,700 M\u207b\u00b9 cm\u207b\u00b9 (Orange G at 477 nm), 22,900 M\u207b\u00b9 cm\u207b\u00b9 (Ponceau BS at 502 nm) [9,10], and 33,470 M\u207b\u00b9 cm\u207b\u00b9 (Ponceau S at 520 nm). One unit (U) of enzyme activity was defined as the amount of enzyme required to degrade 1 \u00b5M azo dye per min. Menadione reductase activity was assayed by employing a similar procedure as above using a molar absorption coefficient of 5060 M\u207b\u00b9 cm\u207b\u00b9 (NADH at 342 nm). Proteins were quantified using the bicinchoninic acid assay (Pierce) with bovine serum albumin (BSA) as the standard. Enzyme purification Proteins were purified at 4 \u00b0C by using an AKTApurifier 10 system with UNICORN 4.10 software (Amersham Biosciences). A single colony of E. coli BL21-Gold(DE3)pLysS harboring pAZOA grown on LB\u2013ampicillin\u2013chloramphenicol plate media was inoculated into a flask containing 50 ml LB\u2013ampicillin\u2013chloramphenicol (50 \u00b5g/ml of each antibiotic) broth. The culture was shaken (250 rpm) at 37 \u00b0C overnight. The next morning, the culture was added to 1 liter LB broth containing no selection antibiotics. The culture was shaken at 250 rpm at 37 \u00b0C for 2 h. Isopropyl-1-thio-\u03b2-galactopyranoside (IPTG, 1 mM) was added and the culture was incubated for another 2.5 h. Cells were harvested by centrifugation (5000g, 10 min), washed with 50 ml buffer containing 25 mM Tris\u2013HCl (pH 7.5), and resuspended in 80 ml of the same buffer. The cells were then disrupted by freezing and thawing followed by 5 min sonication at 4 \u00b0C with a Vibracell VCX 400 model sonifier (Sonics and Materials). Cell debris was removed by centrifugation at 12,000g for 10 min. Forty milliliters of the supernatant (399.6 mg protein) was applied to a HiPrep 16/10 Q XL anion-exchange column (1.6 \u00d7 10 cm, Amersham Biosciences) equilibrated with the same buffer at a linear flow rate of 120 ml/h. Elution with 100 ml of the starting buffer at the same flow rate removed unbound proteins. This was followed by elution with 240 ml of a NaCl gradient from 0 to 1.0 M with a fraction size of 3 ml in the same buffer. Peak fractions of azoreductase activity eluted around 0.25 M NaCl (20 ml) were pooled and concentrated to a volume of about 8 ml by using an Amicon Ultra-15 (Millipore). The sample was dialyzed and buffered with 25 mM Tris\u2013HCl (pH 8.0). The sample was then applied in 2 ml portions to a Mono Q HR 5/5 column (0.5 \u00d7 5 cm, Amersham Biosciences) equilibrated with 25 mM Tris\u2013HCl (pH 8.0) buffer at a linear flow rate of 0.5 ml/min. The column was washed with 5 ml of the starting buffer and eluted with 10 ml of a NaCl gradient from 0 to 1.0 M in the same buffer at the same flow rate. Fractions of 0.5 ml were collected and those containing major enzyme activity were pooled (1.0 ml, around 0.35 M NaCl). Final purification was achieved by gel filtration on a HiLoad 16/60 Superdex 75 pre grade column (1.6 \u00d7 60 cm, Amersham Biosciences) equilibrated and run with 25 mM potassium phosphate (pH 7.1), containing 0.1 M NaCl at a linear flow rate of 1 ml/min. The enzyme solution in 1 ml portions was injected into the column through a sample loop. Fractions of 2 ml were collected, and those containing major enzyme activity were combined (6 ml) and stored at \u201320 \u00b0C. BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) (Amersham Biosciences) were used as the standards. Preparation of apo-enzyme One volume of 10 mM potassium phosphate (pH 7.1) containing 2.0 M ammonium sulfate was mixed with one volume of the purified dimeric enzyme (5 mg) from above. Five milliliters of the sample was applied to a HiPrep 16/10 Phenyl FF hydrophobic interaction column (1.6 \u00d7 10 cm, Amersham Biosciences) equilibrated with the same buffer plus 1.0 M ammonium sulfate at a linear flow rate of 2 ml/min. Four hundred milliliters of a linear gradient of ammonium sulfate from 1.0 to 0 M in the same buffer was applied at the same flow rate. This was followed by further elution with 100 ml of the buffer. The fractions (30 ml) containing apo-enzyme were concentrated and stored at \u201320 \u00b0C. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis and N-terminal amino acid sequencing of the protein Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS\u2013PAGE) was carried out in Laemmli\u2019s buffer [27] with 12.5% polyacrylamide. Perfect protein markers (Novagen) were used. Electrophoresis was performed in a Hoefer SE 260 Mighty Small II Mini Vertical Unit (Amersham Biosciences). Gels were stained for proteins with Coomassie brilliant blue R-250 (Bio-Rad). Amino acid sequencing was done with protein bands from SDS\u2013PAGE. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane in a Hoefer TE77 Semi-Dry Transfer Unit (Amersham Biosciences). The transferred proteins were visualized by Coomassie staining and excised with a razor blade. N-terminal amino acid sequencing was performed on an Applied Biosystems model 477 A gas-phase sequencer equipped with an automatic on-line phenylthiohydantoin analyzer. Apparent kinetic constants of the purified AzoA Initial velocities of the enzymatic reaction were performed by varying the concentrations of one substrate, Methyl red (from 0.005 to 0.04 mM) or NADH (from 0.05 to 0.4 mM), while the concentration of the other substrate was kept constant (NADH: 0.4 mM or Methyl red: 0.04 mM). Apparent Km and Vmax values were obtained from Lineweaver\u2013Burk plots. Nucleotide sequence accession number The nucleotide sequence of azoA of E. faecalis ATCC 19433 has been assigned the GenBank Accession No. AY422207.",
    "LLM": "Materials and methods Bacterial strains, plasmids, and growth conditions Enterococcus faecalis ATCC 19433 was grown in brain heart infusion (BHI) broth or on BHI agar plates and used for inoculum and genomic DNA preparation. E. coli TOP10F\u0001 (Invitrogen), NovaBlue (DE3) (Nova\ufffegen), and BL21-Gold(DE3)pLysS (Stratagene) were used for recombinant DNA studies. E. coli strains were cultured at 37 C in Luria\u2013Bertani (LB) medium with appropriate antibiotics (50 lg/ml). The plasmids pCR2.1-TOPO (Invitrogen) and pET-11a (Stratagene) were used for cloning and expression, respectively. Culture conditions for E. faecalis to grow in various azo dyes Enterococcus faecalis from a BHI agar plate was in\ufffeoculated into 20 ml BHI medium supplemented with individual azo dye at a final concentration of 0.2 mM and incubated at 37 C for 20 h without shaking in air. The cells were harvested by centrifugation at 5000g. The supernatants were used to assay the azo dye reduction by measuring residual absorption at the appropriate wavelength for each azo dye, as described in the enzyme assay description below. The cells were suspended in 1 ml of 25 mM sodium phosphate buffer, pH 7.1, and disrupted mechanically with sea-sand using a Vortex Gene2 (Scientific Industries) for 15 min at 4 C. The mixtures were than centrifuged at 14,000g for 10 min at 4 C and the supernatant fraction was collected and used as cell-free extract. Cloning of E. faecalis azoA gene and expression of AzoA in E. coli Genomic DNA of E. faecalis was isolated essentially as described by Wang et al. [26]. Plasmids from E. coli Top10F\u0001 and NovaBlue (DE3) were isolated using a Qiaprep Spin Miniprep kit (Qiagen). A DNA fragment containing the putative E. faecalis azoA was obtained by PCR with the genomic DNA of E. faecalis used as template. The forward primer included a NdeI site before the start codon: 50 -gattcatatgtcaaaattattagttg-30 (Efs-forward). The reverse primer included a BamHI site downstream of azoA: 50 -cgggatccgctttatgccgcagc taag-30 (Efs-reverse). PCR was performed in a Mas\ufffetercycler gradient (Eppendorf). The amplification con\ufffeditions were one cycle of 95 C for 3 min, 30 cycles with each cycle including 30 s of melting at 95 C, 30 s of annealing at 50 C, and 60 s of extension at 72 C, and one final extension cycle at 72 C for 5 min. The PCR products were examined by 1% agarose gel electro\ufffephoresis. The PCR products amplified from the genomic DNA were directly cloned into pCR2.1-TOPO vector and sequenced. For over-expression of native AzoA in E. coli, the amplified PCR products were cleaved with NdeI and BamHI (New England BioLabs). The digested DNA was purified from the agarose gel and ligated into pET-11a with a rapid DNA ligation kit (Roche). E. coli NovaBlue (DE3) was transformed with the resulting plasmids. The plasmids (pAZOA) were subsequently isolated and introduced into E. coli BL21- Gold(DE3)pLysS by transformation. DNA sequence analysis, translation, and alignment with related genes and proteins were carried out using the Lasergene program (Version 5, DNASTAR). The GenBank program BLAST was utilized to find similar genes or proteins. Enzyme assays Azoreductase activity was assayed by measuring the decrease in optical density at suitable wavelengths with a Hewlett\u2013Packard 8453 UV\u2013visible spectrophotometer at room temperature (23 C). A typical reaction mixture (2.0 ml) contained 25 mM potassium phosphate buffer (pH 7.1), 25 lM azo dye, 0.1 mM NADH, 10 lM FMN, and a suitable amount of enzyme. The reaction was ini\ufffetiated by addition of the enzyme. Initial velocity was de\ufffetermined by monitoring the change in the amount of substrate in the first 2 min in a glass cuvette of 1.0-cm light path. The following molar absorption coefficients were used: 23,360 M\u00011 cm\u00011 (Methyl red at 430 nm), 18,200 M\u00011 cm\u00011 (Orange II at 482 nm), 22,600 M\u00011 cm\u00011 (Amaranth at 520 nm), 20,700 M\u00011 cm\u00011 (Orange G at 477 nm), 22,900 M\u00011 cm\u00011 (Ponceau BS at 502 nm) [9,10], and 33,470 M\u00011 cm\u00011 (Ponceau S at 520 nm). One unit (U) of enzyme activity was defined as the amount of en\ufffezyme required to degrade 1 lM azo dye per min. Mena\ufffedione reductase activity was assayed by employing a similar procedure as above using a molar absorption co\ufffeefficient of 5060 M\u00011 cm\u00011 (NADH at 342 nm). Proteins were quantified using the bicinchoninic acid assay (Pierce) with bovine serum albumin (BSA) as the standard. Enzyme purification Proteins were purified at )4 C by using an AKTApurifier 10 system with UNICORN 4.10 software (Amersham Biosciences). A single colony of E. coli BL21- Gold(DE3)pLysS harboring pAZOA grown on LB\u2013am\ufffepicillin\u2013chloramphenicol plate media was inoculated into a flask containing 50 ml LB\u2013ampicillin\u2013chloramphenicol (50 lg/ml of each antibiotic) broth. The culture was shaken (250 rpm) at 37 C overnight. The next morning, the culture was added to 1 liter LB broth containing no selection antibiotics. The culture was shaken at 250 rpm at 37 C for 2 h. Isopropyl-1-thio-b-galactopyranoside (IPTG, 1 mM) was added and the culture was incubated for another 2.5 h. Cells were harvested by centrifugation (5000g, 10 min), washed with 50 ml buffer containing 25 mM Tris\u2013HCl (pH 7.5), and resuspended in 80 ml of the same buffer. The cells were then disrupted by freezing and thawing followed by 5 min sonication at 4 C with a Vibracell VCX 400 model sonifier (Sonics and Materials). Cell debris was removed by centrifugation at 12,000g for 10 min. Forty milliliters of the supernatant (399.6 mg protein) was applied to a HiPrep 16/10 Q XL anion\ufffeexchange column (1.6 10 cm, Amersham Biosciences) equilibrated with the same buffer at a linear flow rate of 120 ml/h. Elution with 100 ml of the starting buffer at the same flow rate removed unbound proteins. This was fol\ufffelowed by elution with 240 ml of a NaCl gradient from 0 to1.0 M with a fraction size of 3 ml in the same buffer. Peak fractions of azoreductase activity eluted around 0.25 M NaCl (20 ml) were pooled and concentrated to a volume of about 8 ml by using an Amicon Ultra-15 (Millipore). The sample was dialyzed and buffered with 25 mM Tris\u2013HCl (pH 8.0). The sample was then applied in 2 ml portions to a Mono Q HR 5/5 column (0.5 5 cm, Amersham Biosciences) equilibrated with 25 mM Tris\u2013 HCl (pH 8.0) buffer at a linear flow rate of 0.5 ml/min. The column was washed with 5 ml of the starting buffer and eluted with 10 ml of a NaCl gradient from 0 to1.0 M in the same buffer at the same flow rate. Fractions of 0.5 ml were collected and those containing major enzyme activity were pooled (1.0 ml, around 0.35 M NaCl). Final purifi\ufffecation was achieved by gel filtration on a HiLoad 16/60 Superdex 75 pre grade column (1.6 60 cm, Amersham Biosciences) equilibrated and run with 25 mM potassium phosphate (pH 7.1), containing 0.1 M NaCl at a linear flow rate of 1 ml/min. The enzyme solution in 1 ml por\ufffetions was injected into the column through a sample loop. Fractions of 2 ml were collected, and those containing major enzyme activity were combined (6 ml) and stored at )20 C. BSA (67 kDa), ovalbumin (43 kDa), chymo\ufffetrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) (Amersham Biosciences) were used as the standards. Preparation of apo-enzyme One volume of 10 mM potassium phosphate (pH 7.1) containing 2.0 M ammonium sulfate was mixed with one volume of the purified dimeric enzyme (5 mg) from above. Five milliliters of the sample was applied to a HiPrep 16/10 Phenyl FF hydrophobic interaction col\ufffeumn (1.6 10 cm, Amersham Biosciences) equilibrated with the same buffer plus 1.0 M ammonium sulfate at a linear flow rate of 2 ml/min. Four hundred milliliters of a linear gradient of ammonium sulfate from 1.0 to 0 M in the same buffer was applied at the same flow rate. This was followed by further elution with 100 ml of the buf\ufffefer. The fractions (30 ml) containing apo-enzyme were concentrated and stored at )20 C. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis and N-terminal amino acid sequencing of the protein Sodium dodecyl sulfate\u2013polyacrylamide gel electro\ufffephoresis (SDS\u2013PAGE) was carried out in Laemmli\u0001s buffer [27] with 12.5% polyacrylamide. Perfect protein markers (Novagen) were used. Electrophoresis was per\ufffeformed in a Hoefer SE 260 Mighty Small II Mini Vertical Unit (Amersham Biosciences). Gels were stained for proteins with Coomassie brilliant blue R-250 (Bio-Rad). Amino acid sequencing was done with protein bands from SDS\u2013PAGE. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane in a Hoefer TE77 Semi-Dry Transfer Unit (Amersham Biosciences). The transferred proteins were visualized by Coomassie staining and excised with a razor blade. N-terminal amino acid sequencing was performed on an Applied Biosystems model 477 A gas-phase sequencer equipped with an automatic on-line phenylthiohydantoin analyzer. Apparent kinetic constants of the purified AzoA Initial velocities of the enzymatic reaction were per\ufffeformed by varying the concentrations of one substrate, Methyl red (from 0.005 to 0.04 mM) or NADH (from 0.05 to 0.4 mM), while the concentration of the other substrate was kept constant (NADH: 0.4 mM or Methyl red: 0.04 mM). Apparent Km and Vmax values were ob\ufffetained from Lineweaver\u2013Burk plots. Nucleotide sequence accession number The nucleotide sequence of azoA of E. faecalis ATCC 19433 has been assigned the GenBank Accession No. AY422207."
  },
  "21890348": {
    "bleu": 75.43859649122807,
    "meteor": 89.8231272695835,
    "rouge1_p": 0.7711058263971462,
    "rouge1_r": 0.9536764705882353,
    "rouge1_f": 0.8527284681130836,
    "rouge2_p": 0.735871505056514,
    "rouge2_r": 0.9102281089036056,
    "rouge2_f": 0.8138157894736842,
    "rougeL_p": 0.7675386444708681,
    "rougeL_r": 0.9492647058823529,
    "rougeL_f": 0.8487836949375411,
    "cosine_similarity": 0.7504379848886423,
    "precision": 0.565,
    "recall": 0.875968992248062,
    "ner_f1": 0.6869300911854103,
    "ner_tp": 113,
    "ner_fp": 87,
    "ner_fn": 16,
    "bertscore_p": 0.9180406928062439,
    "bertscore_r": 0.9411009550094604,
    "bertscore_f1": 0.9297860264778137,
    "bertscore_scibert_error": "The expanded size of the tensor (2472) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2472].  Tensor sizes: [1, 512]",
    "len_gt": 8101,
    "len_jl": 9652,
    "GT": "2. Methods 2.1. Chemicals All chemicals were of the highest grade available commercially. The dyes tested are listed in Table 1. Three different model wastewaters, designed in the frame of the European Commission (EC) project SOPHIED, to mimic effluents produced during wool and cotton textile dyeing processes were prepared as follows: acid bath (for wool) with AY49, AB62, AR266, AB210, and AB194 dyes at 0.1 g L\u207b\u00b9 each and 2 g L\u207b\u00b9 of Na\u2082SO\u2084, pH 5, reactive bath (for cotton) with RB222, RR195, RY145, and RB5 dyes at 1.25 g L\u207b\u00b9 each and 70 g L\u207b\u00b9 of Na\u2082SO\u2084, pH 10 and direct bath (for cotton) with DB71, DR80, and DY106 dyes at 1 g L\u207b\u00b9 each and 5 g L\u207b\u00b9 of NaCl, pH 9 (Prigione et al., 2008a). All solutions were sterilized by tyndallization (three 1 h-cycles at 80 \u00b0C with 24 h interval between cycles at room temperature) before use. 2.2. Construction of an E. coli strain co-expressing cotA and ppAzoR The ppAzoR gene was PCR amplified from chromosomal DNA of strain P. putida MET94 using the primers PpaF (5\u2032 GCAGGGAATTGCATATGAAACTGTTGCACATCGATTCG 3\u2032) and PpaR (5\u2032 CCATAACCTAGGTCAGGCAGCCGCAAACAGCTCGCTGGC 3\u2032). The resulting 612-bp DNA fragment was purified, digested with NdeI and AvrII and cloned between the same restriction sites of the plasmid pETDuet-1\u2122 (Novagen) to produce pAIF-1. Similarly, for the amplification of cotA gene oligonucleotides CotA159D (5\u2032 CCAGACAAGGACTAGTAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGACACTTGAAAAATTTGTGGATGC 3\u2032) and CotA1892R (5\u2032 CGCGGATCCTTTATGGGGATCAGTTATATCC 3\u2032) were used. The product of the reaction (1539-bp) was digested with SpeI and BamHI purified and cloned into pAIF-1 previously digested with XbaI and BamHI, to yield pAIF-2. The correct sequence of the inserts was confirmed by sequencing. The plasmid pAIF-2 was introduced into the host expression strain E. coli BL21star (DE3), producing the strain LOM529, in which both genes were expressed under the control of the T7lac promoter. 2.3. Overproduction of enzymes in heterologous host The recombinant strain LOM529 was grown in Luria Bertani (LB) medium supplemented with ampicillin (100 \u00b5g mL\u207b\u00b9) at 37 \u00b0C. Growth was followed until the midlog phase (OD600 = 0.6), at which time 0.1 mM isopropyl-\u03b2-D-thiogalactopyranoside (IPTG) and 0.25 mM CuCl\u2082 were added to the culture medium and the temperature lowered to 25 \u00b0C. Incubation and agitation continued for a further 4 h, after which the agitation was stopped and the growth remained static overnight at 25 \u00b0C. Cells were harvested by centrifugation (8000 \u00d7 g, 10 min, 4 \u00b0C). To measure the enzymatic activity in crude extracts, cell suspensions were prepared in 20 mM phosphate buffer, pH 8, containing DNase I (10 \u00b5g mL\u207b\u00b9 extract), MgCl\u2082 (5 mM), and a mixture of protease inhibitors, antipain and leupeptin (2 \u00b5g mL\u207b\u00b9 extract). Cells were disrupted in a French Press and cell debris was removed by centrifugation (18,000 \u00d7 g, 2 h, 4 \u00b0C). Supernatants were used for SDS\u2013PAGE analysis and enzymatic assays. Laccase activity was determined using ABTS as substrate (Martins et al., 2002). Azoreductase activity was measured using NADPH and reactive black 5 azo dye as substrates as described before (Mendes et al., 2011). 2.4. Enzyme assays using purified enzymes The recombinant PpAzoR from P. putida and the recombinant CotA-laccase from B. subtilis were produced and purified as previously described (Martins et al., 2002; Mendes et al., 2011). For the PpAzoR reactions serum bottles contained 0.25 mM NADPH and 3 mM of dye (or 0.5 mM for toxicity studies) in Britton Robinson (BR) buffer (0.1 M phosphoric acid, 0.1 M boric acid, 0.1 M acetic acid titrated to the desired pH with 0.5 M NaOH) pH 7, at 30 \u00b0C. The bottles were sealed with rubber stoppers and made anaerobic by argon bubbling. Reactions were initiated by the injection of anoxic-made purified enzyme through the use of a syringe. The CotA-laccase reactions were assessed using 3 mM of dye (or 0.5 mM for toxicity studies) in BR buffer pH 8, 37 \u00b0C. Samples were withdrawn at different times, diluted, and the absorbance measured at the maximal wavelength of the dyes tested using a discontinuous assay on a Nicolet Evolution 300 spectrophotometer (Thermo Industries). One unit (U) of enzymatic activity was defined as the amount of enzyme required to reduce 1 \u00b5mol of the substrate per minute. The molar absorbance of dyes is listed at Table 1. All enzymatic assays were performed at least in triplicate. 2.5. Enzyme assays using recombinant whole cells The recombinant E. coli cells overproducing PpAzoR and CotA were harvested at the late-exponential phase of growth by centrifugation (8,000 \u00d7 g, 10 min, 4 \u00b0C) and washed twice with a 0.9% (w/v) NaCl solution. Cells were permeabilized before use, by shaking at 250 rpm 30 \u00b0C during 30 min upon addition of toluene (0.1% v/v) or Triton X-100 (0.1% v/v). The permeabilization level was assessed by measuring protein content and activity in cells supernatants. For the enzymatic assays, the washed pellet was suspended in 20 mM phosphate buffer, pH 8 to an OD600 = 20, unless otherwise stated. The cells were added to a sealed serum bottle containing 5 mL of each model wastewater bath, or 5 mL of 0.5 mM of RB5 in phosphate buffer, pH 8, at 30 \u00b0C. The reaction mixtures were previously degassed with purified argon, providing the appropriate anoxic conditions for PpAzoR catalysis. Samples were withdrawn at different times and the decolourization was monitored spectrophotometrically at three different wavelengths (400, 500 and 600 nm) after appropriate dilution for the model wastewater baths, or at 600 nm for reactive black 5. After this period of time the reaction mixtures were made aerobic through shaking at 150 rpm and the temperature was increased to 37 \u00b0C; these aerobic conditions inhibited PpAzoR activity while promoting CotA laccase oxidative action over dyestuff. 2.6. Toxicity analysis Toxicity assays were performed based on the inhibitory effects of dyes and products of enzymatic degradation on the growth of the yeast S. cerevisiae BY4741 (Papaefthimiou et al., 2004; Pereira et al., 2009a,b), or on the reproduction of the nematode C. elegans wild type strain Bristol N2 (Anderson et al., 2001). Briefly, the yeast cell suspension used as inoculum was prepared from a mid-exponential phase culture, which was centrifuged and suspended to OD640nm = 0.15 in a triple strength minimal growth medium that contained (per litre): 1.7 g YNB without aminoacids or NH\u2084\u207a (Difco, Detroit, USA), 20 g glucose (Merck, Germany), 2.65 g (NH\u2084)\u2082SO\u2084 (Merck), 20 mg methionine, 20 mg histidine, 60 mg leucine and 20 mg uracil (Sigma, USA). For the toxicity tests, 50 \u00b5l of the standardized yeast cell suspension were mixed with 100 \u00b5l of test or control solutions in 96-well polystyrene microplates (Greiner Bio-one) that were sealed and incubated at 30 \u00b0C for 16 h with constant agitation. Growth of the yeast cell population was assessed by measuring the optical density (OD750nm, which did not interfere with any of the maximal absorption wavelength of the dyes tested) attained after 16 h of incubation. The toxicity was estimated based on the percentage of yeast growth inhibition defined as 1 \u2013 (ODx750nm / OD0750nm) \u00d7 100 where ODx750nm and OD0750nm are the absorbance values attained by the yeast cell population in the presence and in the absence of each test solution, respectively. For the reproduction assays with C. elegans Bristol N2, experiments were performed in 48-well polystyrene microplates (Greiner Bio-one) by adding a single worm from age-synchronized cultures at the L4 larval stage, to 50 \u00b5l of 10\u00d7 K medium (Anderson et al., 2001), 50 \u00b5l of E. coli OP50 suspension with an OD640nm = 1.5 and 400 \u00b5l of test or control solutions. The plates were incubated at 20 \u00b0C in a FITOCLIMA S600 (Aralab) incubator. After incubation for 96 h, the number of offspring at all beyond the eggs was scored, using a Zeiss Stemi 2000-C stereomicroscope (50\u00d7 magnification). Data reported are average values with standard deviations of results from at least two independent toxicity experiments performed in quadruplicate.",
    "LLM": "2. Methods 2.1. Chemicals All chemicals were of the highest grade available commercially. The dyes tested are listed in Table 1. Three different model waste\u00adwaters, designed in the frame of the European Commission (EC) project SOPHIED, to mimic effluents produced during wool and cotton textile dyeing processes were prepared as follows: acid bath (for wool) with AY49, AB62, AR266, AB210, and AB194 dyes at 0.1 g L1 each and 2 g L1 of Na2SO4, pH 5, reactive bath (for cot\u00ad ton) with RB222, RR195, RY145, and RB5 dyes at 1.25 g L1 each and 70 g L1 of Na2SO4, pH 10 and direct bath (for cotton) with DB71, DR80, and DY106 dyes at 1 g L1 each and 5 g L1 of NaCl, pH 9 (Prigione et al., 2008a). All solutions were sterilized by tyn\u00ad dallization (three 1 h-cycles at 80 C with 24 h interval between cycles at room temperature) before use. 2.2. Construction of an E. coli strain co-expressing cotA and ppAzoR The ppAzoR gene was PCR amplified from chromosomal DNA of strain P. putida MET94 using the primers PpaF (50 GCAGGGA\u00ad ATTGCATATGAAACTGTTGCACATCGATTCG 30 ) and PpaR (50 CCAT AACCTAGGTCAGGCAGCCGCAAACAGCTCGCTGGC 30 ). The resulting 612-bp DNA fragment was purified, digested with NdeI and AvrII and cloned between the same restriction sites of the plasmid pETD\u00ad uet-1TM (Novagen) to produce pAIF-1. Similarly, for the amplifica\u00ad tion of cotA gene oligonucleotides CotA159D (50 CCAGACAAGGA CTAGTAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGACACTT GAAAAATTTGTGGATGC 30 ) and CotA1892R (50 CGCGGATCCTT\u00ad TATGGGGATCAGTTATATCC 30 ) were used. The product of the reac\u00ad tion (1539-bp) was digested with SpeI and BamHI purified and cloned into pAIF-1 previously digested with XbaI and BamHI, to yield pAIF-2. The correct sequence of the inserts was confirmed by sequencing. The plasmid pAIF-2 was introduced into the host expression strain E. coli BL21star (DE3), producing the strain LOM529, in which both genes were expressed under the control of the T7lac promoter. 2.3. Overproduction of enzymes in heterologous host The recombinant strain LOM529 was grown in Luria Bertani (LB) medium supplemented with ampicillin (100 gmL1 ) at 37 C. Growth was followed until the midlog phase (OD600 = 0.6), at which time 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and 0.25 mM CuCl2 were added to the culture medium and the Table 1 Color index (C.I.) generic names, C.I registration numbers, absorption maxima, purity and calculated molar extinction coefficients of dyes used in this study a . C.I. generic name C.I. constitution number Dye content (%) Absorption max (nm) Direct Blue 1 (DB1) 24,410 80 610 Direct Red R (DRR) 22,120 91 530 Direct Black 38 (DB38) 30,235 50 600 Direct Blue 71 (DB71) 34,140 NSb 565 Direct Red 80 (DR80) 35,780 NS 555 Direct Yellow 106 (DY106) 1332 50 420 Reactive Red 4 (RR4) 18,105 50 530 Reactive Black 5 (RB5) 20,505 55 600 Reactive Yellow 145 (RY145) c 50 420 Reactive Blue 222 (RB222) c NS 600 Reactive Red 195 (RR195) c 30 550 Acid Red 299 (AR299) c NS 440 Acid Black 210 (AB210) 300,285 30 600 Acid Yellow 49 (AY49) 18,640 50 390 Acid Black 194 (AB194) c 60 570 Acid Red 266 (AR266) 17,101 30 470 Acid Blue 62 (AB62) 62,045 100 600 Acid Orange 7 (AO7) 15,510 20\u201330 480 Sudan Orange G (SOG) 11,920 98 430 Methyl Red (MR) 11,920 95 430 Mordant Black 3 (MB3) 14,640 30 550 Mordant Black 9 (MB9) 14,855 60\u201385 550 Mordant Black 17 (MB17) 15,705 50 530 a The dyes were purchased from Sigma\u2013Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany), Town End (Leeds, UK), DyStar Textilfarben (Germany), Yorkshire Europe (Belgium) and Bezema AG (Montlinglen, Switzerland). The absorption maxima were determined in BR buffer (pH 7). If the dye purity was not indicated by the supplier, it was assumed that the preparations consisted of pure dye. b NS, dye purity not specified by the supplier. C Confidential. temperature lowered to 25 C. Incubation and agitation continued for a further 4 h, after which the agitation was stopped and the growth remained static overnight at 25 C. Cells were harvested by centrifugation (8000 \u0004 g, 10 min, 4 C). To measure the enzy\u00ad matic activity in crude extracts, cell suspensions were prepared in 20 mM phosphate buffer, pH 8, containing DNase I (10 lg mL1 extract), MgCl2 (5 mM), and a mixture of protease inhibitors, anti\u00ad pain and leupeptin (2 lg mL1 extract). Cells were disrupted in a French Press and cell debris was removed by centrifugation (18000 \u0004 g, 2 h, 4 C). Supernatants were used for SDS\u2013PAGE anal\u00ad ysis and enzymatic assays. Laccase activity was determined using ABTS as substrate (Martins et al., 2002). Azoreductase activity was measured using NADPH and reactive black 5 azo dye as sub\u00ad strates as described before (Mendes et al., 2011). 2.4. Enzyme assays using purified enzymes The recombinant PpAzoR from P. putida and the recombinant CotA-laccase from B. subtilis were produced and purified as previ\u00ad ously described (Martins et al., 2002; Mendes et al., 2011). For the PpAzoR reactions serum bottles contained 0.25 mM NADPH and 3 mM of dye (or 0.5 mM for toxicity studies) in Britton Robin\u00ad son (BR) buffer (0.1 M phosphoric acid, 0.1 M boric acid, 0.1 M ace\u00ad tic acid titrated to the desired pH with 0.5 M NaOH) pH 7, at 30 C. The bottles were sealed with rubber stoppers and made anaerobic by argon bubbling. Reactions were initiated by the injection of an\u00ad oxic-made purified enzyme through the use of a syringe. The CotA\u00ad laccase reactions were assessed using 3 mM of dye (or 0.5 mM for toxicity studies) in BR buffer pH 8, 37 C. Samples were withdrawn at different times, diluted, and the absorbance measured at the maximal wavelength of the dyes tested using a discontinuous as\u00ad say on a Nicolet Evolution 300 spectrophotometer (Thermo Indus\u00ad tries). One unit (U) of enzymatic activity was defined as the amount of enzyme required to reduce 1 lmol of the substrate per minute. The molar absorbance of dyes is listed at Table 1. All enzymatic assays were performed at least in triplicate. 2.5. Enzyme assays using recombinant whole cells The recombinant E. coli cells overproducing PpAzoR and CotA were harvested at the late-exponential phase of growth by centri\u00ad fugation (8,000 \u0004 g, 10 min, 4 C) and washed twice with a 0.9% (w/v) NaCl solution. Cells were permeabilized before use, by shaking at 250 rpm 30 C during 30 min upon addition of toluene (0.1% v/v) or Triton X-100 (0.1% v/v). The permeabilization level was assessed by measuring protein content and activity in cells supernatants. For the enzymatic assays, the washed pellet was sus\u00ad pended in 20 mM phosphate buffer, pH 8 to an OD600 = 20, unless otherwise stated. The cells were added to a sealed serum bottle containing 5 mL of each model wastewater bath, or 5 mL of 0.5 mM of RB5 in phosphate buffer, pH 8, at 30 C. The reaction mixtures were previously degassed with purified argon, providing the appropriate anoxic conditions for PpAzoR catalysis. Samples were withdrawn at different times and the decolourization was monitored spectrophotometrically at three different wavelengths (400, 500 and 600 nm) after appropriate dilution for the model wastewater baths, or at 600 nm for reactive black 5. After this per\u00ad iod of time the reaction mixtures were made aerobic through shak\u00ad ing at 150 rpm and the temperature was increased to 37 C; these aerobic conditions inhibited PpAzoR activity while promoting CotA laccase oxidative action over dyestuff. 2.6. Toxicity analysis Toxicity assays were performed based on the inhibitory effects of dyes and products of enzymatic degradation on the growth of the yeast S. cerevisiae BY4741 (Papaefthimiou et al., 2004; Pereira et al., 2009a,b), or on the reproduction of the nematode C. elegans wild type strain Bristol N2 (Anderson et al., 2001). Briefly, the yeast cell suspension used as inoculum was prepared from a mid-exponential phase culture, which was centrifuged and sus\u00ad pended to OD640nm = 0.15 in a triple strength minimal growth medium that contained (per litre): 1.7 g YNB without aminoacids or NH4 + (Difco, Detroit, USA), 20 g glucose (Merck, Germany), 2.65 g (NH4)2SO4 (Merck), 20 mg methionine, 20 mg histidine, 60 mg leucine and 20 mg uracil (Sigma, USA). For the toxicity tests, 50 l of the standardized yeast cell suspension were mixed with 100 l of test or control solutions in 96-well polystyrene micro\u00ad plates (Greiner Bio-one) that were sealed and incubated at 30 C for 16 h with constant agitation. Growth of the yeast cell popula\u00ad tion was assessed by measuring the optical density (OD750nm, which did not interfere with any of the maximal absorption wave\u00ad length of the dyes tested) attained after 16 h of incubation. The toxicity was estimated based on the percentage of yeast growth inhibition defined as 1ODX 750nm OD0 750nm \u0004 100 where ODX 750nm and OD0 750nm are the absorbance values attained by the yeast cell population in the presence and in the absence of each test solution, respec\u00ad tively. For the reproduction assays with C. elegans Bristol N2, experiments were performed in 48-well polystyrene microplates (Greiner Bio-one) by adding a single worm from age-synchronized cultures at the L4 larval stage, to 50 l of 10 \u0004 K medium (Anderson et al., 2001), 50 l of E. coli OP50 suspension with an OD640nm = 1.5 and 400 l of test or control solutions. The plates were incubated at 20 C in a FITOCLIMA S600 (Aralab) incubator. After incubation for 96 h, the number of offspring at all beyond the eggs was scored, using a Zeiss Stemi 2000-C stereomicroscope (50 \u0004 magnification). Data reported are average values with standard deviations of re\u00ad sults from at least two independent toxicity experiments per\u00ad formed in quadruplicate."
  },
  "17904577": {
    "bleu": 82.13939980638915,
    "meteor": 83.57505315440548,
    "rouge1_p": 0.8179347826086957,
    "rouge1_r": 0.9868852459016394,
    "rouge1_f": 0.8945022288261516,
    "rouge2_p": 0.7971723762914628,
    "rouge2_r": 0.9619422572178478,
    "rouge2_f": 0.871840618495391,
    "rougeL_p": 0.816304347826087,
    "rougeL_r": 0.9849180327868853,
    "rougeL_f": 0.8927191679049034,
    "cosine_similarity": 0.8442419265059603,
    "precision": 0.7486631016042781,
    "recall": 0.9032258064516129,
    "ner_f1": 0.8187134502923976,
    "ner_tp": 140,
    "ner_fp": 47,
    "ner_fn": 15,
    "bertscore_p": 0.6786183714866638,
    "bertscore_r": 0.6770958304405212,
    "bertscore_f1": 0.6791511178016663,
    "bertscore_scibert_error": "The expanded size of the tensor (2897) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2897].  Tensor sizes: [1, 512]",
    "len_gt": 9450,
    "len_jl": 11055,
    "GT": "Materials and Methods Materials Chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Reagents for Molecular Biology were purchased from Promega, DNA primers for gene amplifications were synthesized by Sigma-Aldrich. The pH of buffer solutions was adjusted at the appropriate temperature. Bacterial strains, plasmid and media Bacterial strain P. aeruginosa PAO1 was kindly provided by Dr Gail Preston, Department of Plant Sciences, Oxford University, UK. All bacteria cultures were grown in Luria-Bertani (LB) liquid medium or LB agar plate with additional supplements stated. The pET28b(+) plasmid vector (Novagen), E. coli JM109 and E. coli BL21(DE3)pLysS (Promega) were used for DNA and recombinant expression work. For selection of positive cloning outcomes, suitable antibiotics were supplemented according to manufacturer's instructions. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from a 6 ml culture of P. aeruginosa PAO1 using RNeasy (Qiagen). The cDNA was synthesized from 1 \u03bcg of total RNA template using the ThermoScript RT-PCR system (Invitrogen) and applied as a template for gene-specific primed PCR amplification using following primers: PA0785 forward (NdeI) (5\u2032 gatacatATGagtagaattcttgcagtgc 3\u2032), reverse (XhoI) (5\u2032 tggcctcgagTCAggccgaccgcgccagcg 3\u2032), PA1962 forward (NdeI) (5\u2032 gagcgacatATGaaacttttgcatatcg 3\u2032), reverse (EcoRI) (5\u2032 gaatTCAggccgcggcgaactgcccgg 3\u2032), PA3223 forward (NdeI) (5\u2032 gatacatATGtcccgtgtcctggttatgg 3\u2032), reverse (EagI) (5\u2032 cccggccgtcacaccggcaaccat 3\u2032), with the restriction enzyme recognition sites underlined and start/stop codons in capital letters. The amplification mixtures (2 mM Mg2+, 200 \u03bcM dNTPs each, 6% (v/v) dimethyl sulfoxide (DMSO), 1 \u03bcM each primer, 1 \u03bcg genomic DNA, 2.5 units Pfu DNA polymerase (Promega) in a total 100 \u03bcl) were subjected to the following thermo-cycling protocol: one cycle of 95 \u00b0C denaturation for 2 min, 30 cycles of elongation (1 min of melting at 95 \u00b0C, 30 s of annealing at 60 \u00b0C, and 1 min 30 s of extension at 72 \u00b0C), an extra cycle of extension at 72 \u00b0C for 10 min, and a final cooling cycle at 4 \u00b0C for 5 min. The PCR products were separated by gel electrophoresis in ethidium bromide-stained agarose (1.0%, w/v). Cloning of PA0785 from P. aeruginosa PAO1 The gDNA isolated from of P. aeruginosa PAO1 was used as the template for the amplification of the gene PA0785 by PCR reaction described above. PCR products were purified from 1.0% (w/v) agarose gel electrophoresis with the QIAquick Gel Extraction Kit (Qiagen) and restriction digested followed by ligation into pET28b(+) plasmid vector, generating an PA0785-containing expression vector. Chemically competent E. coli JM109 were transformed with the PA0785-containing vector, and the transformed constructs were verified by sequencing analysis. The correct plasmid constructs were subsequently transformed into host expression strain E. coli BL21(DE3)pLysS by the heat shock method. Heterologous overexpression and purification of recombinant PA0785 Recombinant paAzoR1 was produced in LB-sorbitol (LB containing 1 M sorbitol and 2.5 mM betaine) supplemented with 30 \u03bcg/ml of kanamycin (180 rpm, 37 \u00b0C and 25 \u00b0C after induction with 0.5 mM isopropyl-\u03b2-D-thiogalactopyranoside (IPTG) when cell optical density reached 0.4\u20130.6). Bacterial culture was supplemented with 0.1 mM FMN as additional source of flavin co-factor. The hexahistidine tag of the purified recombinant paAzoR1 was cleaved by thrombin digestion (2 units per mg protein, 16 h at 4 \u00b0C) and removed by using a 10 kDa cut-off protein concentrator (Amicon Ultra-15; Millipore). Pure recombinant protein was stored at \u221280 \u00b0C in the presence of 5% (v/v) glycerol. Determination of protein concentration The protein content of bacterial lysate and eluates was estimated using the Bradford assay with bovine serum albumin (BSA) as standard. The concentration of purified recombinant azoreductase was determined by measuring absorption at 280 nm with an extinction coefficient of 15,470 M\u22121 cm\u22121, as calculated by ExPASy ProtParam.55 Gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli,56 with slight modifications for native PAGE (12% polyacrylamide gel; Bio-Rad), i.e. SDS and \u03b2-mercaptoethanol were omitted. The gel solution was prepared in 25 mM Tris\u2013HCl (pH 8.0) with no added NaCl. Polyacrylamide gels were stained with Coomassie blue R-250. Protein marker: low-range molecular mass protein markers (Bio-Rad), were used as standards for SDS\u2013PAGE. Size exclusion chromatography Size-exclusion gel filtration was carried out on a Sephacryl S-200 (Amersham Biosciences) XK16/60 column (Pharmacia), connected to an \u00c4KTA Purifier system (Amersham Pharmacia Biotech). The column was equilibrated with 20 mM Tris\u2013HCl (pH 8.0) containing 0.3 M NaCl. A 200 \u03bcl sample containing 0.8 mg purified paAzoR1 was loaded onto the column and eluted with buffer described above at a flow rate of 0.5 ml/min at 4 \u00b0C. The eluate was monitored at wavelengths of 280 and 460 nm. Molecular mass standards applied were: thyroglobulin (670 kDa), catalase (250 kDa), alcohol dehydrogenase (141 kDa), BSA (66 kDa) and trypsin inhibitor (21.5 kDa), all at 5 mg/ml. Enzymic activity assays and substrate profiles The activity of purified recombinant paAzoR1 was determined by following the decrease in absorbance due to the reductive cleavage of the coloured azo substrates. Decrease of optical density of each azo compound was measured with a U-2001 spectrophotometer (Hitachi) at the following wavelengths: amaranth (519 nm), ponceau S (511 nm), ponceaus BS (505 nm), orange II (485 nm), orange G (478 nm), tropaeolin (447 nm), methyl red (435 nm), 4-aminoazobenzene-4\u2032-sulfonic acid (PAABSA), sulfasalazine, balsalazide, and olsalazine (405 nm). The enzymatic reaction was initiated by adding 0.5 mM NAD(P)H to a mixture of (50 \u03bcM azo substrate, 10 \u03bcg enzyme, 20 mM Tris\u2013HCl (pH 8.0), 0.3 M NaCl). The final reaction volume was 200 \u03bcl. Initial velocities of enzymic reaction were obtained by varying the concentrations of one substrate, azo compounds (from 5 \u03bcM \u2013200 \u03bcM) or NAD(P)H (from 0.1 mM\u20131.0 mM), while the concentration of the other substrate kept constant. Apparent Vmax and Km values were determined by non-linear regression with the program KyPlot. Thermostability of paAzoR1 was measured with methyl red as substrate in the assay described above, with the enzyme pre-incubated for 10 min at temperatures ranging from 4 \u00b0C\u201390 \u00b0C and then equilibrated for 1 min on ice. The acyl carrier protein phosphodiesterase activity was measured as described, and the acyl carrier protein phosphodiesterase from E. coli was used as a positive control. Spectral properties of paAzoR1 Denaturation of paAzoR1 was achieved by incubating enzyme with 1% (w/v) SDS for 15 min. \u03b2-NADPH was supplied as reducing agents for FMN. Flavin standard was dissolved in 20 mM Tris\u2013HCl (pH 8.0), 0.3 M NaCl. Spectra were recorded with a U-2001 spectrophotometer (Hitachi) across the wavelength of 635 nm to 300 nm. Bioinformatics and sequence alignment Amino acid sequences were identified by BLAST search of the database by using the protein sequence of the azoreductase from E. coli JM109 as the query. Peptide sequences were downloaded from Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). Sequences were aligned with CLUSTAL W, and Figure 1 was generated with Espript 2.2. X-ray structure determination The pure recombinant paAzoR1 protein was concentrated to 23 mg/ml with Amicon ultracentrifugation concentrators (Millipore, Watford, UK). Crystals of paAzoR1 with the substrate methyl red were grown at 19 \u00b0C by the sitting-drop vapour diffusion method. Equal volumes (0.15 \u03bcl) of protein solution (23 mg/ml in 20 mM Tris\u2013HCl buffer (pH 8.0) with 2 mM methyl red) were mixed with mother liquor (Molecular Dimensions Screen II condition 14: 1.6 M (NH4)2SO4, 0.1 M Hepes (pH 7.5)) by using a Mosquito crystallisation robot (TTP Labtech, Royston, UK). Crystals of approximate dimensions 30 \u03bcm \u00d7 30 \u03bcm \u00d7 30 \u03bcm grew typically in two to five days. Crystals were briefly transferred to a cryo-protectant solution of 1 : 3 glycerol/mother liquor prior to snap-freezing in liquid nitrogen. Data were collected at beamlines ID29 and ID14-eh3 at the European Synchrotron Radiation Facility (ESRF, Grenoble) with a Quantum ADSC detector. Data were integrated with iMosflm v0.5.2, and scaled and merged with SCALA. The Laue group was determined with Pointless, and discrimination between the two most likely space groups (P31 21 and P32 21) was made following molecular replacement with the program PHASER. An ensemble of proteins (RCSB Protein Data Bank accession codes 1V4B and 1T5B) was used for molecular replacement. Iterative model building and refinement was performed with Coot and Refmac 5.3. Initial translation, libration and screw (TLS) parameters were determined with the program TLSMD, and TLS refinement was performed within Refmac. Tight non-crystallographic symmetry (NCS) restraints were applied to the two protein chains in the asymmetric unit. Water molecules were added with Coot and with Arp/Warp. A final refinement step was performed in PHENIX. Model validation was performed with PROCHECK and molprobity, and multimer analysis was performed by using PISA. The data collection and refinement statistics are shown in Table 4.",
    "LLM": "Materials and Methods Materials Chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Reagents for Molecular Biology were purchased from Promega, DNA primers for gene amplifications were synthesized by Sigma-Aldrich. The pH of buffer solutions was adjusted at the appropriate temperature. Bacterial strains, plasmid and media Bacterial strain P. aeruginosa PAO1 was kindly pro- vided by Dr Gail Preston, Department of Plant Sciences, Oxford University, UK. All bacteria cultures were grown in Luria-Bertani (LB) liquid medium or LB agar plate with additional supplements stated. The pET28b(+) plasmid vector (Novagen), E. coli JM109 and E. coli BL21(DE3)pLysS (Promega) were used for DNA and recombinant expression work. For selection of positive cloning outcomes, suitable antibiotics were supplemen- ted according to manufacturer's instructions. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from a 6 ml culture of P. aeruginosa PAO1 using RNeasy (Qiagen). The cDNA was synthesized from 1 \u03bcg of total RNA template using the ThermoScript RT-PCR system (Invitrogen) and applied as a template for gene-specific primed PCR amplification using following primers: PA0785 forward (NdeI) (5\u2032 gatacatATGagtagaattcttgcagtgc 3\u2032), reverse (XhoI) (5\u2032 tggcctcgagTCAggccgaccgcgccagcg 3\u2032), PA1962 forward (NdeI) (5\u2032 gagcgacatATGaaacttttgcatatcg 3\u2032), reverse (EcoRI) (5\u2032 gaatTCAggccgcggcgaactgcccgg 3\u2032), PA3223 forward (NdeI) (5\u2032 gatacatATGtcccgtgtcctggttatgg 3\u2032), reverse (EagI) (5\u2032 cccggccgtcacaccggcaaccat 3\u2032), with the restriction enzyme recognition sites underlined and start/ stop codons in capital letters. The amplification mixtures (2 mM Mg2+, 200 \u03bcM dNTPs each, 6% (v/v) dimethyl sulfoxide (DMSO), 1 \u03bcM each primer, 1 \u03bcg genomic DNA, 2.5 units Pfu DNA polymerase (Promega) in a total 100 \u03bcl) were subjected to the following thermo-cycling protocol: one cycle of 95 \u00b0C denaturation for 2 min, 30 cycles of elongation (1 min of melting at 95 \u00b0C, 30 s of annealing at 60 \u00b0C, and 1 min 30 s of extension at 72 \u00b0C), an extra cycle of extension at 72 \u00b0C for 10 min, and a final cooling cycle at 4 \u00b0C for 5 min. The PCR products were separated by gel electrophoresis in ethidium bromide- stained agarose (1.0%, w/v). Cloning of PA0785 from P. aeruginosa PAO1 The gDNA isolated from of P. aeruginosa PAO1 was used as the template for the amplification of the gene PA0785 by PCR reaction described above. PCR products were purified from 1.0% (w/v) agarose gel electrophoresis with the QIAquick Gel Extraction Kit (Qiagen) and restriction digested followed by ligation into pET28b(+) plasmid vector, generating an PA0785-containing expres- sion vector. Chemically competent E. coli JM109 were transformed with the PA0785-containing vector, and the transformed constructs were verified by sequencing analysis. The correct plasmid constructs were subse- quently transformed into host expression strain E. coli BL21(DE3)pLysS by the heat shock method. Heterologous overexpression and purification of recombinant PA0785 Recombinant paAzoR1 was produced in LB-sorbitol (LB containing 1 M sorbitol and 2.5 mM betaine) supplemented with 30 \u03bcg/ml of kanamycin (180 rpm, 37 \u00b0C and 25 \u00b0C after induction with 0.5 mM isopropyl-\u03b2- D-thiogalactopyranoside (IPTG) when cell optical density reached 0.4\u20130.6). Bacterial culture was supplemented with 0.1 mM FMN as additional source of flavin co-factor. The hexahistidine tag of the purified recombinant paAzoR1 was cleaved by thrombin digestion (2 units per mg protein, 16 h at 4 \u00b0C) and removed by using a 10 kDa cut-off protein concentrator (Amicon Ultra-15; Millipore). Pure recombinant protein was stored at \u221280 \u00b0C in the presence of 5% (v/v) glycerol. Determination of protein concentration The protein content of bacterial lysate and eluates was estimated using the Bradford assay with bovine serum albumin (BSA) as standard. The concentration of purified recombinant azoreductase was determined by measuring absorption at 280 nm with an extinction coefficient of 15,470 M\u22121 cm\u22121 , as calculated by ExPASy ProtParam.55 Gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) was per- formed by the method of Laemmli,56 with slight modifica- tions for native PAGE (12% polyacrylamide gel; Bio-Rad), i.e. SDS and \u03b2-mercaptoethanol were omitted. The gel solution was prepared in 25 mM Tris\u2013HCl (pH 8.0) with no added NaCl. Polyacrylamide gels were stained with Coomassie blue R-250. Protein marker: low-range mole- cular mass protein markers (Bio-Rad), were used as standards for SDS\u2013PAGE. Size exclusion chromatography Size-exclusion gel filtration was carried out on a Sephacryl S-200 (Amersham Biosciences) XK16/60 col- umn (Pharmacia), connected to an \u00c4KTA Purifier system (Amersham Pharmacia Biotech). The column was equili- brated with 20 mM Tris\u2013HCl (pH 8.0) containing 0.3 M NaCl. A 200 \u03bcl sample containing 0.8 mg purified paAzoR1 was loaded onto the column and eluted with buffer described above at a flow rate of 0.5 ml/min at 4 \u00b0C. The eluate was monitored at wavelengths of 280 and 460 nm. Molecular mass standards applied were: thyr- oglobulin (670 kDa), catalase (250 kDa), alcohol dehydro- genase (141 kDa), BSA (66 kDa) and trypsin inhibitor (21.5 kDa), all at 5 mg/ml. Enzymic activity assays and substrate profiles The activity of purified recombinant paAzoR1 was determined by following the decrease in absorbance due to the reductive cleavage of the coloured azo substrates.4 Decrease of optical density of each azo compound was measured with a U-2001 spectrophotometer (Hitachi) at the following wavelengths: amaranth (519 nm), ponceau S (511 nm), ponceaus BS (505 nm), orange II (485 nm), orange G (478 nm), tropaeolin (447 nm), methyl red (435 nm), 4-aminoazobenzene-4\u2032-sulfonic acid (PAABSA), sulfasalazine, balsalazide, and olsalazine (405 nm). The enzymatic reaction was initiated by adding 0.5 mM NAD(P)H to a mixture of (50 \u03bcM azo substrate, 10 \u03bcg enzyme, 20 mM Tris\u2013HCl (pH 8.0), 0.3 M NaCl). The final reaction volume was 200 \u03bcl. Initial velocities of enzymic reaction were obtained by varying the concen- trations of one substrate, azo compounds (from 5 \u03bcM \u2013 200 \u03bcM) or NAD(P)H (from 0.1 mM\u20131.0 mM), while the concentration of the other substrate kept constant. Apparent Vmax and Km values were determined by non-linear regression with the program KyPlot\u00a7. Ther- mostability of paAzoR1 was measured with methyl red as substrate in the assay described above, with the enzyme pre-incubated for 10 min at temperatures ranging from 4 \u00b0C\u201390 \u00b0C and then equilibrated for 1 min on ice. The acyl carrier protein phosphodiesterase activity was measured as described, and the acyl carrier protein phosphodiesterase from E. coli was used as a positive control.37,57 Spectral properties of paAzoR1 Denaturation of paAzoR1 was achieved by incubat- ing enzyme with 1% (w/v) SDS for 15 min. \u03b2-NADPH was supplied as reducing agents for FMN. Flavin standard was dissolved in 20 mM Tris\u2013HCl (pH 8.0), 0.3 M NaCl. Spectra were recorded with a U-2001 spectrophotometer (Hitachi) across the wavelength of 635 nm to 300 nm. Bioinformatics and sequence alignment Amino acid sequences were identified by BLAST search\u2225 of the database by using the protein sequence of the azoreductase from E. coli JM109 as the query. Peptide sequences were downloaded from Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). Sequences were aligned with CLUSTAL W\u00b6,58 and Figure 1 was generated with Espript 2.2a . 59 X-ray structure determination The pure recombinant paAzoR1 protein was concen- trated to 23 mg/ml with Amicon ultracentrifugation concentrators (Millipore, Watford, UK). Crystals of paA- zoR1 with the substrate methyl red were grown at 19 \u00b0C by the sitting-drop vapour diffusion method. Equal volumes (0.15 \u03bcl) of protein solution (23 mg/ml in 20 mM Tris\u2013HCl buffer (pH 8.0) with 2 mM methyl red) were mixed with mother liquor (Molecular Dimensions Screen II condition 14: 1.6 M (NH4)2SO4, 0.1 M Hepes (pH 7.5)) by using a Mosquito crystallisation robot (TTP Labtech, Royston, UK). Crystals of approximate dimen- sions 30 \u03bcm \u00d7 30\u03bcm \u00d7 30 \u03bcm grew typically in two to five days. Crystals were briefly transferred to a cryo-protectant solution of 1 : 3 glycerol/mother liquor prior to snap- freezing in liquid nitrogen. Data were collected at beamlines ID29 and ID14-eh3 at the European Synchro- tron Radiation Facility (ESRF, Grenoble) with a Quantum ADSC detector. Data were integrated with iMosflm v0.5.2,60 and scaled and merged with SCALA.61 The Laue group was determined with Pointless,61 and discrimination between the two most likely space groups (P31 2 1 and P32 2 1) was made following molecular replacement with the program PHASER.62 An ensemble of proteins (RCSB Protein Data Bank accession codes 1V4B and 1T5B) was used for molecular replacement. Iterative model building and refinement was performed with Coot63 and Refmac 5.3.64 Initial translation, libration and screw (TLS) parameters were determined with the program TLSMD,65 and TLS refinement was performed within Refmac. Tight non-crystallographic symmetry (NCS) restraints were applied to the two protein chains in the asymmetric unit. Water molecules were added with Coot and with Arp/Warp.66 A final refinement step was performed in PHENIX.67 Model validation was performed with PROCHECK68 and molprobity,69 and multimer analysis was performed by using PISA.b70 The data collection and refinement statistics are shown in Table 4. Protein Data Bank accession code The structure of paAzoR1 has been deposited at the RCSB Protein Data Bank (PDB accession code: 2v9c). \u00a7 http://www.woundedmoon.org/win32/kyplot.html \u2225 http://www.ncbi.nlm.nih.gov/BLAST/ Table 4. Crystal parameters, X-ray data collection and refinement statistics Space group P31 2 1 Cell dimensions a, b, c (\u00c5) 82.55, 82.55, 108.65 \u03b1, \u03b2, \u03b3 (\u00b0) 90, 90, 120 Data collection statistics Wavelength (\u00c5) 0.97630 Resolution (\u00c5) 34.0\u20132.18 (2.30\u20132.18)a No. unique reflections 20,681 (2462) Rsymb 0.055 (0.46) I/\u03c3(I) 9.7 (1.6) Completeness (%) 97.4 (88.8) Redundancy 3.5 (2.3) Refinement and model statistics Resolution (\u00c5) 34.0\u20132.18 (2.23\u20132.18) No. reflections used (working set) 38,938 Rworkc 19.4 (32.7) Rfreec 24.2 (34.4) Number of residues (chain A/B) 203/193 Number of water molecules 171 Additional molecules 3 \u00d7 glycerol Total number of atoms 3364 rmsd bond lengths (\u00c5) 0.017 rmsd bond angles (\u00b0) 0.98 Mean B factor 43.3 Ramachandran statistics (%) Core region 92.5 Additional allowed region 7.5 Generously allowed 0.0 Disallowed 0.0 a Numbers in the parentheses are for the highest resolution shell. b Rsym \u00bc P h P j jIh;j hIhij P h P j Ih;j ; where Ih,j is the intensity of the jth observation of unique reflection h. c Rwork and Rfree \u00bc P hjjFohjjFchjj P hjFohj for the working set and test set (5%) of reflections, where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h. \u00b6 http://www.ebi.ac.uk/clustalw/ a http://espript.ibcp.fr/ESPript/ESPript/"
  },
  "20417637": {
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    "rouge1_p": 0.9937304075235109,
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    "rougeL_p": 0.9937304075235109,
    "rougeL_r": 0.9723926380368099,
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    "cosine_similarity": 0.9608304668653808,
    "precision": 0.9411764705882353,
    "recall": 0.9142857142857143,
    "ner_f1": 0.9275362318840579,
    "ner_tp": 64,
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    "bertscore_p": 0.969413697719574,
    "bertscore_r": 0.9573841094970703,
    "bertscore_f1": 0.9635289907455444,
    "bertscore_scibert_error": "The expanded size of the tensor (992) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 992].  Tensor sizes: [1, 512]",
    "len_gt": 4099,
    "len_jl": 3997,
    "GT": "Materials and Methods Materials Chemicals were purchased from Sigma-Aldrich, unless otherwise stated. DNA primers were also synthesized by Sigma-Aldrich. Molecular biology, bacterial strains, and protein production The P. aeruginosa PAO1 Washington strain was kindly provided by Dr. Gail Preston (Department of Plant Sciences, University of Oxford, Oxford, UK). The primers used in the PCR amplifications of paazor2 and paazor3 are summarised in Supplementary Table 2. All bacterial cultures were grown on LB liquid medium or on LB agar plate, with the additional supplements stated. The pET28b(+) plasmid vector (Novagen), E. coli JM109, and E. coli BL21(DE3)pLysS (Promega) were used for DNA and recombinant expression work. For selection of positive cloning outcomes, antibiotics were supplemented as follows, where appropriate: 30 \u03bcg mL\u22121 kanamycin and 34 \u03bcg mL\u22121 chloramphenicol, both final concentrations. Cloning procedures, expression, and purification of recombinant paAzoR2 and paAzoR3, and their enzymic characterisations were conducted as previously described for paAzoR1.13 The reduction of 2,6-dichloroindophenol was monitored via the decrease in absorbance at 600 nm. Protein quantification The concentration of recombinant paAzoR2 and paAzoR3 was determined by measuring absorption at 280 nm with extinction coefficients of 18,450 M\u22121 cm\u22121, 22,585 M\u22121 cm\u22121, and 13,980 M\u22121 cm\u22121, respectively, as calculated by ExPASy ProtParam.66,67 Protein crystallization and structural determination The crystals of paAzoR1 complexed with balsalazide were grown by sitting-drop vapour diffusion. paAzoR1 plus balsalazide protein drops were prepared by mixing equal volumes (0.2 \u03bcl) of protein solution [23 mg mL\u22121 in 20 mM Tris\u2013HCl (pH 8.0) with 2 mM balsalazide] with precipitant solution [Molecular Dimensions JCSG-plus screen condition B11: 0.2 M lithium sulfate, 0.1 M Tris\u2013HCl (pH 8.5), and 30% wt/vol polyethylene glycol 4000] by using a Mosquito crystallisation robot (TTP Labtech). Crystals were briefly transferred to a cryoprotectant solution of 1:3 glycerol/precipitant solution prior to snap freezing in liquid nitrogen. Data for paAzoR1_BLZ were collected at beamline I02 at the Diamond Light Source (Oxon, UK) with a Quantum ADSC detector. Data were integrated with iMosflm v0.5.2,68 and scaled and merged with SCALA.69 The structure was solved with the program PHASER,70 using the paAzoR1 structure (PDB code 2V9C) as search model. The atomic model was rebuilt and refined with Coot,71 Refmac 5.4,72 and PHENIX.73 Initial TLS (translation, liberation, and screw) parameters were determined with the program TLSMD,74 and TLS refinement was performed with PHENIX. The noncrystallographic symmetry restraints (tight main chain and moderate side chain) were applied to the two protein chains in the asymmetric unit. Water molecules were added with PHENIX, Coot, and ARP/wARP.75 Model validation was performed with MolProbity,35 and multimer analysis was performed using PISA.76 Data collection and refinement statistics are shown in Table 3. Homology modeling of paAzoR2 and paAzoR3 To help determine the reasons for the differences in substrate specificity among the azoreductases, we generated homology models for paAzoR2 and paAzoR3 using the comparative modeling program Modeller (version 9v7).77 The models were created using a template based on the structures of azoreductases from four bacteria: E. coli (PDB code 2Z9B),53 Sal. typhimurium (PDB code 1T5B),78 P. aeruginosa (PDB code 2V9C),13 and En. faecalis (PDB code 2HPV).41 The template was also based on the sequence alignment shown in Fig. 1. Four structures were used to create the template due to the low sequence identities between the azoreductases and paAzoR2 and paAzoR3 (all <45% identical). The models for paAzoR2 and paAzoR3 were superimposed onto the structure of E. coli azoreductase (RMSD \u223c0.6 \u00c5) using Swiss-PdbViewer (v4.0.1),79 and FMN was positioned within the models based on that superposition. Accession numbers Coordinates and structure factors have been deposited in the PDB with accession number 3LT5.",
    "LLM": "Materials and Methods Materials Chemicals were purchased from Sigma-Aldrich, unless otherwise stated. DNA primers were also synthesized by Sigma-Aldrich. Molecular biology, bacterial strains, and protein production The P. aeruginosa PAO1 Washington strain was kindly provided by Dr. Gail Preston (Department of Plant Sciences, University of Oxford, Oxford, UK). The primers used in the PCR amplifications of paazor2 and paazor3 are summarised in Supplementary Table 2. All bacterial cultures were grown on LB liquid medium or on LB agar plate, with the additional supplements stated. The pET28b(+) plasmid vector (Novagen), E. coli JM109, and E. coli BL21(DE3)pLysS (Promega) were used for DNA and recombinant expression work. For selection of positive cloning outcomes, antibiotics were supplemen\ufffeted as follows, where appropriate: 30 \u03bcg mL\u2212 1 kanamycin and 34 \u03bcg mL\u22121 chloramphenicol, both final concentrations. Cloning procedures, expression, and purification of recombinant paAzoR2 and paAzoR3, and their enzymic characterisations were conducted as previously described for paAzoR1.13 The reduction of 2,6- dichloroindophenol was monitored via the decrease in absorbance at 600 nm. Protein quantification The concentration of recombinant paAzoR2 and paAzoR3 was determined by measuring absorption at 280 nm with extinction coefficients of 18,450 M\u22121 cm\u22121 , 22,585 M\u22121 cm\u22121 , and 13,980 M\u22121 cm\u22121 , respectively, as calculated by ExPASy ProtParam.66,67 Protein crystallization and structural determination The crystals of paAzoR1 complexed with balsalazide were grown by sitting-drop vapour diffusion. paAzoR1 plus balsalazide protein drops were prepared by mixing equal volumes (0.2 \u03bc l) of protein solution [23 mg mL\u22121 in 20 mM Tris\u2013HCl (pH 8.0) with 2 mM balsalazide] with precipitant solution [Molecular Dimensions JCSG-plus screen condition B11: 0.2 M lithium sulfate, 0.1 M Tris\u2013 HCl (pH 8.5), and 30% wt/vol polyethylene glycol 4000] by using a Mosquito crystallisation robot (TTP Labtech). Crystals were briefly transferred to a cryoprotectant solution of 1:3 glycerol/precipitant solution prior to snap freezing in liquid nitrogen. Data for paAzoR1_BLZ were collected at beamline I02 at the Diamond Light Source (Oxon, UK) with a Quantum ADSC detector. Data were integrated with iMosflm v0.5.2,68 and scaled and merged with SCALA.69 The structure was solved with the program PHASER,70 using the paAzoR1 structure (PDB code 2V9C) as search model. The atomic model was rebuilt and refined with Coot,71 Refmac 5.4,72 and PHENIX.73 Initial TLS (translation, liberation, and screw) parameters were determined with the program TLSMD,74 and TLS refinement was performed with PHENIX. The noncrystallographic symmetry restraints (tight main chain and moderate side chain) were applied to the two protein chains in the asymmetric unit. Water molecules were added with PHENIX, Coot, and ARP/wARP.75 Model validation was performed with MolProbity,35 and multimer analysis was performed using PISA.76 Data collection and refinement statistics are shown in Table 3. Homology modeling of paAzoR2 and paAzoR3 To help determine the reasons for the differences in substrate specificity among the azoreductases, we gener\ufffeated homology models for paAzoR2 and paAzoR3 using the comparative modeling program Modeller (version 9v7).77 The models were created using a template based on the structures of azoreductases from four bacteria: E. coli (PDB code 2Z9B),53 Sal. typhimurium (PDB code 1T5B),78 P. aeruginosa (PDB code 2V9C),13 and En. faecalis (PDB code 2HPV).41 The template was also based on the sequence alignment shown in Fig. 1. Four structures were used to create the template due to the low sequence identities between the azoreductases and paAzoR2 and paAzoR3 (all b45% identical). The models for paAzoR2 and paAzoR3 were superimposed onto the structure of E. coli azoreductase (RMSD \u223c0.6 \u00c5) using Swiss-PdbViewer (v4.0.1),79 and FMN was positioned within the models based on that superposition."
  },
  "2202258": {
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    "cosine_similarity": 0.9645151529369345,
    "precision": 0.9404761904761905,
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    "ner_f1": 0.9518072289156626,
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    "bertscore_p": 0.98118656873703,
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    "bertscore_f1": 0.9837284088134766,
    "bertscore_scibert_error": "The expanded size of the tensor (1567) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1567].  Tensor sizes: [1, 512]",
    "len_gt": 7281,
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    "GT": "MATERIALS AND METHODS Media and culture conditions. The medium used for the isolation of azoreductase-producing bacteria from intestinal microbial flora was brain heart infusion (BHI; Difco Laboratories, Detroit, Mich.) prepared in accordance with manufacturer instructions and supplemented with the following ingredients per liter: Bacto-Agar (Difco), 2 g; yeast extract, 0.5 g; vitamin K1, 1 mg; hemin, 5 mg; cysteine, 0.5 g; and either Direct Blue 15 (3,3'-dimethoxybenzidine-based dye) or Nitro Red [4-amino-5-hydroxy-3-(4-nitrophenylazo)-2,7-naphthalenedisulfonic acid disodium salt], 80 mg (Fig. 1). The medium used for the growth and maintenance of the azoreductase-producing bacteria was BHI broth (prereduced and anaerobically sterilized; Carr-Scarborough Microbiologicals, Stone Mountain, Ga.). M10 medium was prepared as described by Caldwell and Bryant (2), except that the volatile fatty acids were replaced by acetic acid and propionic acid. Isolation of azoreductase-producing bacteria. Azoreductase-producing bacteria were isolated from human intestinal microflora as follows. One gram of freshly voided human feces was diluted in 9 ml of BHI broth under anaerobic gas (5% CO2, 10% hydrogen, 85% nitrogen). Tenfold serial dilutions of the sample were made in BHI broth, and 0.1-ml dilutions of 10^-5 to 10^-9 were plated on BHI agar plates containing Direct Blue 15 or Nitro Red (final concentration, 80 mg/ml). The plates were incubated under both aerobic and anaerobic conditions at 37\u00b0C and observed for clearance of the dye surrounding the colonies. Approximately 20% of the colonies developed clear zones after plating of the dilutions of fecal samples from the same source at different times. The colonies which cleared the dye were transferred to BHI agar plates containing Direct Blue 15 for single-colony isolation. The isolated colonies were transferred to BHI broth for growth and maintenance. Identification of azoreductase-producing anaerobic bacteria. The anaerobic bacteria capable of reducing Direct Blue 15 were identified by the methods described by Holdeman et al. (11) and in Bergey's Manual of Systematic Bacteriology (14, 27) with media from Carr-Scarborough Microbiologicals. The identification of the isolates was verified by comparing the morphology, staining characteristics, results of biochemical tests, and cellular proteins with those of known cultures of bacteria from the American Type Culture Collection, Rockville, Md. Proteins were compared by gel electrophoresis in polyacrylamide gels by the methods described by Moore et al. (22). Kinetics of degradation of azo dyes by different isolates. The bacterial isolates which could reduce Direct Blue 15 under anaerobic conditions were used for this experiment. To overnight cultures grown in BHI broth, 50 \u03bcmol of sterile Direct Blue 15 was added per ml. The cultures were incubated at 37\u00b0C. Two milliliters from each of five replicate cultures was removed aseptically under anaerobic conditions at 2-h intervals. The samples were centrifuged at 15,000 \u00d7 g for 5 min in tightly capped tubes to ensure anaerobiosis. The supernatant was used to measure the disappearance of the dye over time with a DU-7 spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.) at a wavelength of 615 nm, the lambda max for Direct Blue 15 (3), and with water for a blank. Enzymatic activity in the supernatants of the isolates. Overnight cultures of the isolates grown in BHI broth were centrifuged at 15,000 \u00d7 g for 15 min in tightly capped tubes to ensure anaerobiosis. The supernatants were transferred to tubes containing 2 ml of heavy mineral oil under anaerobic conditions for the assay of enzymatic activity. Direct Blue 15 and tetracycline were added to the supernatants to final concentrations of 50 to 100 \u03bcM and 15 \u03bcg/ml, respectively, and the tubes were incubated at 37\u00b0C. For a control, BHI broth with Direct Blue 15 was used. The cultures were observed and monitored for a decrease in the A615 until the dye was completely cleared and reduced, at which point the absorbance reached a constant level. The time required for total reduction of the dye was noted. The amount of soluble protein from each strain was measured in the supernatants prior to incubation with the dye. The pellets from these cultures were used for bacterial counting. Protein determination and bacterial counting. The amount of soluble protein produced by each bacterium in the spent medium after incubation (which was used for the determination of enzymatic activity) was determined by the method of Lowry et al. (17) with bovine serum albumin as a standard. The amount of protein in the uninoculated medium was small and was subtracted from the value obtained for soluble protein from each strain in the spent medium. Bacterial counting (in the pellets from the cultures used for the determination of enzymatic activity) was performed by plating serial dilutions of bacteria on BHI agar plates. Effects of cofactors on the azoreductase activity of the isolates. M10 medium was used for this experiment and was prepared as either broth or agar medium. Flavin adenine dinucleotide (FAD), flavin mononucleotide, or riboflavin (final concentration, 50 \u03bcg/ml) and Direct Blue 15 (final concentration, 25 \u03bcg/ml) were added to the medium. Only the dye was added to the control medium. The bacteria from a single colony of either the new isolates or Clostridium perfringens were used as inocula. Cultures were inoculated under anaerobic conditions and incubated anaerobically at 37\u00b0C. Other compounds tested at a concentration of 50 \u03bcg/ml were NAD, NADH, NADPH, glutathione, and cytochrome c. M10 medium plates containing, in addition to 50 \u03bcg of FAD per ml, mersalyl acid (final concentration, 50 \u03bcg/ml) were also prepared. They were inoculated with C. perfringens or one of the new isolates. Native gel electrophoresis of protein for the detection of azoreductase. The enzyme in culture supernatants was precipitated by the addition of 60% ammonium sulfate, and the precipitate was dissolved in BHI broth. Bromophenol blue was added to the protein as a marker before the sample was loaded on the gel. In some experiments, pelleted cells were broken by sonic oscillation and loaded directly on the gel after the addition of bromophenol blue. The separating gel for nondenaturing polyacrylamide gels contained 10% acrylamide and 0.26% bis and was prepared in accordance with the instructions provided by Sigma Chemical Co., St. Louis, Mo. (technical bulletin no. MKR-137). The stacking gel contained 2.5% acrylamide and 0.5 \u03bcg of riboflavin per ml. The electrode buffer contained 5 mM Tris and 37.3 mM glycine (pH 8.3). Thirty to 50 \u03bcg of each protein sample was used for loading the gel. A model V16 vertical slab gel electrophoresis apparatus (Bethesda Research Laboratories, Gaithersburg, Md.) was used for electrophoresis. A constant current of 6 mA was applied to the gel until the bromophenol blue was close to the anodic end of the gel. All steps for the preparation and running of the gel had to be performed under anaerobic conditions to ensure the activity of the enzyme. At the conclusion of the run, the gel was stained with Nitro Red or Direct Blue 15 and incubated at 37\u00b0C under anaerobic conditions.",
    "LLM": "MATERIALS AND METHODS Media and culture conditions. The medium used for the isolation of azoreductase-producing bacteria from intestinal microbial flora was brain heart infusion (BHI; Difco Laboratories, Detroit, Mich.) prepared in accordance with manufacturer instructions and supplemented with the following ingredients per liter: Bacto-Agar (Difco), 2 g; yeast extract, 0.5 g; vitamin K1, 1 mg; hemin, 5 mg; cysteine, 0.5 g; and either Direct Blue 15 (3,3'-dimethoxybenzidine-based dye) or Nitro Red [4-amino-5-hydroxy-3-(4-nitrophenylazo)-2,7-naphthalenedisulfonic acid disodium salt], 80 mg (Fig. 1). The medium used for the growth and maintenance of the azoreductase-producing bacteria was BHI broth (prereduced and anaerobically sterilized; Carr-Scarborough Microbiologicals, Stone Mountain, Ga.). M10 medium was prepared as described by Caldwell and B ---ant (2), except that the volatile fatty acids were replaced by acetic acid and propionic acid. Isolation of azoreductase-producing bacteria. Azoreductase-producing bacteria were isolated from human intestinal microflora as follows. One gram of freshly voided human feces was diluted in 9 ml of BHI broth under anaerobic gas (5% C02, 10% hydrogen, 85% nitrogen). Tenfold serial dilutions of the sample were made in BHI broth, and 0.1-ml dilutions of i0-5 to 10-9 were plated on BHI agar plates containing Direct Blue 15 or Nitro Red (final concentration, 80 mg/ml). The plates were incubated under both aerobic and anaerobic conditions at 37\u00b0C and observed for clearance of the dye surrounding the colonies. Approximately 20% of the colonies developed clear zones after plating of the dilutions of fecal samples from the same source at different times. The colonies which cleared the dye were transferred to BHI agar plates containing Direct Blue 15 for single-colony isolation. The isolated colonies were transferred to BHI broth for growth and maintenance. Identification of azoreductase-producing anaerobic bacteria. The anaerobic bacteria capable of reducing Direct Blue 15 were identified by the methods described by Holdeman et al. (11) and in Bergey's Manual of Systematic Bacteriology (14, 27) with media from Carr-Scarborough Microbiologicals. The identification of the isolates was verified by comparing the morphology, staining characteristics, results of biochemical tests, and cellular proteins with those of known cultures of bacteria from the American Type Culture Collection, Rockville, Md. Proteins were compared by gel electrophoresis in polyacrylamide gels by the methods described by Moore et al. (22). Kinetics of degradation of azo dyes by different isolates. The bacterial isolates which could reduce Direct Blue 15 under anaerobic conditions were used for this experiment. To overnight cultures grown in BHI broth, 50 ,umol of sterile Direct Blue 15 was added per ml. The cultures were incubated at 37\u00b0C. Two milliliters from each of five replicate cultures was removed aseptically under anaerobic conditions at 2-h intervals. The samples were centrifuged at 15,000 x g for 5 min in tightly capped tubes to ensure anaerobiosis. The supernatant was used to measure the disappearance of the dye over time with a DU-7 spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.) at a wavelength of 615 nm, the lambda max for Direct Blue 15 (3), and with water for a blank. Enzymatic activity in the supernatants of the isolates. Overnight cultures of the isolates grown in BHI broth were centrifuged at 15,000 x g for 15 min in tightly capped tubes to ensure anaerobiosis. The supernatants were transferred to tubes containing 2 ml of heavy mineral oil under anaerobic conditions for the assay of enzymatic activity. Direct Blue 15 and tetracycline were added to the supernatants to final concentrations of 50 to 100 p,M and 15 ,ug/ml, respectively, and the tubes were incubated at 37\u00b0C. For a control, BHI broth with Direct Blue 15 was used. The cultures were observed and monitored for a decrease in the A615 until the dye was completely cleared and reduced, at which point the absorbance reached a constant level. The time required for total reduction of the dye was noted. The amount of soluble protein from each strain was measured in the supernatants prior to incubation with the dye. The pellets from these cultures were used for bacterial counting. Protein determination and bacterial counting. The amount of soluble protein produced by each bacterium in the spent medium after incubation (which was used for the determination of enzymatic activity) was determined by the method of Lowry et al. (17) with bovine serum albumin as a standard. The amount of protein in the uninoculated medium was small and was subtracted from the value obtained for soluble protein from each strain in the spent medium. Bacterial counting (in the pellets from the cultures used for the determination of enzymatic activity) was performed by plating serial dilutions of bacteria on BHI agar plates. Effects of cofactors on the azoreductase activity of the isolates. M10 medium was used for this experiment and was prepared as either broth or agar medium. Flavin adenine dinucleotide (FAD), flavin mononucleotide, or riboflavin (final concentration, 50 p,g/ml) and Direct Blue 15 (final concentration, 25 ,ug/ml) were added to the medium. Only the dye was added to the control medium. The bacteria from a single colony of either the new isolates or Clostridium perfringens were used as inocula. Cultures were inoculated under anaerobic conditions and incubated anaerobically at 37\u00b0C. Other compounds tested at a concentration of 50 ,ug/ml were NAD, NADH, NADPH, glutathione, and cytochrome c. M1O medium plates containing, in addition to 50 p,g of FAD per ml, mersalyl acid (final concentration, 50 ,ug/ml) were also prepared. They were inoculated with C. perfringens or one of the new isolates. Native gel electrophoresis of protein for the detection of azoreductase. The enzyme in culture supernatants was precipitated by the addition of 60% ammonium sulfate, and the precipitate was dissolved in BHI broth. Bromophenol blue was added to the protein as a marker before the sample was loaded on the gel. In some experiments, pelleted cells were broken by sonic oscillation and loaded directly on the gel after the addition of bromophenol blue. The separating gel for nondenaturing polyacrylamide gels contained 10% acrylamide and 0.26% bis and was prepared in accordance with the instructions provided by Sigma Chemical Co., St. Louis, Mo. (technical bulletin no. MKR-137). The stacking gel contained 2.5% acrylamide and 0.5 ,ug of riboflavin per ml. The electrode buffer contained 5 mM Tris and 37.3 mM glycine (pH 8.3). Thirty to 50 ,ug of each protein sample was used for loading the gel. A model V16 vertical slab gel electrophoresis apparatus (Bethesda Research Laboratories, Gaithersburg, Md.) was used for electrophoresis. A constant current of 6 mA was applied to the gel until the bromophenol blue was close to the anodic end of the gel. All steps for the preparation and running of the gel had to be performed under anaerobic conditions to ensure the activity of the enzyme. At the conclusion of the run, the gel was stained with Nitro Red or Direct Blue 15 and incubated at 37\u00b0C under anaerobic conditions."
  },
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    "cosine_similarity": 0.9768201697883769,
    "precision": 0.92,
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    "ner_f1": 0.9154228855721394,
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    "bertscore_p": 0.971146821975708,
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    "bertscore_f1": 0.9727435111999512,
    "bertscore_scibert_error": "The expanded size of the tensor (1271) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1271].  Tensor sizes: [1, 512]",
    "len_gt": 5072,
    "len_jl": 5099,
    "GT": "EXPERIMENTAL All chemicals were obtained from Sigma\u2013Aldrich unless specified. NADH, NADPH and IPTG were purchased from Apollo Scientific, and FMN and FAD were obtained from Tokyo Chemical Industries. Restriction enzymes were purchased from New England Biolabs. Cloning of azoreductases Genomic DNA was extracted from overnight cultures of P. aeruginosa PAO1 grown in LB at 37 \u00b0C using a Gentra Puregene Yeast/Bacteria kit (Qiagen) and was used as a template to amplify the genes pa0949 (paWrbA), pa1204 (paYieF), pa2280 (paArsH) and pa2580 (paMdaB). PCR was performed using a Hotstar Hifidelity Taq kit (Qiagen) and the primers described in Supplementary Table S1. All PCR products were digested using NdeI and SacI prior to cloning into a similarly digested pET-28b plasmid (Novagen). Ligation of the genes into pET-28b was performed using T4 DNA ligase (Promega) and pET-28b including azoreductase genes were transformed into E. coli JM109 (Promega). Transformants were selected on LB agar containing 30 \u03bcg/ml kanamycin. The sequences of all clones were confirmed (Eurofins MWG Biotech) prior to transformation into E. coli BL-21 (Promega). Expression and purification of azoreductases All azoreductase genes were expressed as described for paAzoR1\u20133 [19]. Briefly, E. coli BL-21 cells were grown in LB supplemented with 1 M Sorbitol, 2.5 mM betaine and 30 \u03bcg/ml kanamycin. Cultures were grown to a OD600 of 0.4\u20130.5 and induced with 0.5 mM IPTG for 16 h at 18 \u00b0C. Cultures were centrifuged for 20 min at 6000 g at 4 \u00b0C and supernatant was removed. Cells were then resuspended in lysis buffer containing Complete protease inhibitors (Roche) prior to sonication. The suspension was then centrifuged at 16 000 g to remove debris and the clarified supernatant applied to a Ni\u00b2\u207a-nitrilotriacetate (Ni-NTA) (Invitrogen) column. Protein was eluted via increasing imidazole concentrations in the elution buffer from 0 to 250 mM stepwise. The exception being paYieF which required a 500 mM imidazole wash for complete elution. Prior to storage, all samples containing pure azoreductase were dialysed overnight against 20 mM Tris/HCl, pH 8, and 100 mM NaCl at 4 \u00b0C in 14 kDa cut-off dialysis tubing. Protein concentration and overall yield were determined using the following \u03b5\u2082\u2088\u2080 values obtained using Protparam [24]: paWrbA = 16 055 M\u207b\u00b9\u00b7cm\u207b\u00b9, paYieF = 19 940 M\u207b\u00b9\u00b7cm\u207b\u00b9, paArsH = 29 575 M\u207b\u00b9\u00b7cm\u207b\u00b9 and paMdaB = 39 420 M\u207b\u00b9\u00b7cm\u207b\u00b9. All samples were divided into aliquots and stored at \u201380 \u00b0C in 5% (v/v) glycerol. UV\u2013visible absorbance scans were performed on 100 \u03bcl of 1 mg/ml azoreductase in a 1-ml cuvette using an M200 infinite Pro (Tecan) plate reader. TLC Flavin cofactors were identified via the use of TLC as described previously [25]. Briefly, a mobile phase of 2:1:1 mixture of n-butanol/acetic acid/water was used with a 0.2 mm silica gel-coated plate. Pure stocks of FMN and FAD were spotted as standards for comparison. Plates were visualized under UV illumination. Differential scanning fluorimetry Differential scanning fluorimetry (DSF) was performed via a protocol similar to that described previously [26]. Azoreductases were diluted to 64 \u03bcg/ml in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. The buffer contained Sypro Orange (Invitrogen) and was supplemented with either 1 or 20 \u03bcM FMN or FAD as indicated. Fluorescence measurements were made in an Mx3005p qRT-PCR instrument (Stratagene) and the temperature was increased from 25 \u00b0C to 95 \u00b0C at 1 \u00b0C/min. Data analysis was performed in GraphPad Prism with fitting to a Boltzman sigmoidal function. Enzymatic assays The putative azoreductases were screened against a range of azo and quinone substrates (Supplementary Figure S1). Azoreductase and nitrofurazone reductase assays were performed in a 100 \u03bcl final volume with a reaction mixture as follows: 1.6\u2013100 \u03bcg/ml azoreductase, 50 \u03bcM azo/nitrofuran substrate, 500 \u03bcM NAD(P)H and 1 \u03bcM FMN/FAD in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. The concentration of enzyme was adjusted to maintain a linear rate of reaction for 1 min to allow accurate rate determination. Reactions were monitored at the optimal wavelength for each substrate using an M200 Infinite Pro plate reader (Tecan). Rates were determined over the initial linear portion of the graph. Quinone reductase assays were performed as described previously [14] with the addition of 1 \u03bcM flavin to the reaction buffer. The effect of FMN concentration on enzymatic activity was measured using Phenol Blue (Phb) as the substrate. The reaction mixture was as follows: 0.2\u201310 \u03bcg/ml azoreductase, 100 \u03bcM Phb, 500 \u03bcM NAD(P)H and 1\u201320 \u03bcM FMN/FAD in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. A fixed concentration of each azoreductase was used for assays at all concentrations of flavin. Phb reduction was measured via the change in absorbance at 660 nm. Homology modelling A homology model for paMdaB was generated using the phyre2 web server [27] in intensive mode. The paMdaB model was based upon the structures of MdaB from Yersinia pestis (PDB code 3RPE, 59% identical) and E. coli (PDB code 2AMJ [28], 61% identical).",
    "LLM": "EXPERIMENTAL All chemicals were obtained from Sigma\u2013Aldrich unless specified. NADH, NADPH and IPTG were purchased from Apollo Scientific, and FMN and FAD were obtained from Tokyo Chemical Industries. Restriction enzymes were purchased from New England Biolabs. Cloning of azoreductases Genomic DNA was extracted from overnight cultures of P. aeruginosa PAO1 grown in LB at 37 \u25e6C using a Gentra Puregene Yeast/Bacteria kit (Qiagen) and was used as a template to amplify the genes pa0949 (paWrbA), pa1204 (paYieF), pa2280 (paArsH) and pa2580 (paMdaB). PCR was performed using a Hotstar Hifidelity Taq kit (Qiagen) and the primers described in Supplementary Table S1. All PCR products were digested using NdeI and SacI prior to cloning into a similarly digested pET-28b plasmid (Novagen). Ligation of the genes into pET\ufffe28b was performed using T4 DNA ligase (Promega) and pET-28b including azoreductase genes were transformed intoE. coliJM109 (Promega). Transformants were selected on LB agar containing 30 \u03bcg/ml kanamycin. The sequences of all clones were confirmed (Eurofins MWG Biotech) prior to transformation into E. coli BL-21 (Promega). Expression and purification of azoreductases All azoreductase genes were expressed as described for paAzoR1\u20133 [19]. Briefly, E. coli BL-21 cells were grown in LB supplemented with 1 M Sorbitol, 2.5 mM betaine and 30 \u03bcg/ml kanamycin. Cultures were grown to a OD600 of 0.4\u20130.5 and induced with 0.5 mM IPTG for 16 h at 18 \u25e6C. Cultures were centrifuged for 20 min at 6000 g at 4 \u25e6C and supernatant was removed. Cells were then resuspended in lysis buffer containing Complete protease inhibitors (Roche) prior to sonication. The suspension was then centrifuged at 16 000 g to remove debris and the clarified supernatant applied to a Ni2 + - nitrilotriacetate (Ni-NTA) (Invitrogen) column. Protein was eluted via increasing imidazole concentrations in the elution buffer from 0 to 250 mM stepwise. The exception being paYieF which required a 500 mM imidazole wash for complete elution. Prior to storage, all samples containing pure azoreductase were dialysed overnight against 20 mM Tris/HCl, pH 8, and 100 mM NaCl at 4 \u25e6C in 14 kDa cut-off dialysis tubing. Protein concen\ufffetration and overall yield were determined using the following \u03b5280 values obtained using Protparam [24]: paWrbA = 16 055 M\u2212 1 \u00b7 cm \u2212 1 , paYieF = 19 940 M\u2212 1 \u00b7 cm \u2212 1 , paArsH = 29 575 M\u2212 1 \u00b7 cm \u2212 1 and paMdaB = 39 420 M\u2212 1 \u00b7 cm \u2212 1 . All samples were divided into aliquots and stored at \u201380 \u25e6C in 5% (v/v) glycerol. UV\u2013visible absorbance scans were performed on 100 \u03bcl of 1 mg/ml azoreductase in a 1-ml cuvette using an M200 infinite Pro (Tecan) plate reader. TLC Flavin cofactors were identified via the use of TLC as described previously [25]. Briefly, a mobile phase of 2:1:1 mixture of n\ufffebutanol/acetic acid/water was used with a 0.2 mm silica gel-coated plate. Pure stocks of FMN and FAD were spotted as standards for comparison. Plates were visualized under UV illumination. Differential scanning fluorimetry Differential scanning fluorimetry (DSF) was performed via a protocol similar to that described previously [26]. Azoreductases were diluted to 64 \u03bcg/ml in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. The buffer contained Sypro Orange (Invitrogen) and was supplemented with either 1 or 20 \u03bcM FMN or FAD as indicated. Fluorescence measurements were made in an Mx3005p qRT\ufffePCR instrument (Stratagene) and the temperature was increased from 25 \u25e6C to 95 \u25e6C at 1 \u25e6C/min. Data analysis was performed in GraphPad Prism with fitting to a Boltzman sigmoidal function. Enzymatic assays The putative azoreductases were screened against a range of azo and quinone substrates (Supplementary Figure S1). Azoreductase and nitrofurazone reductase assays were performed in a 100 \u03bcl final volume with a reaction mixture as follows: 1.6\u2013100 \u03bcg/ml azoreductase, 50 \u03bcM azo/nitrofuran substrate, 500 \u03bcM NAD(P)H and 1 \u03bcM FMN/FAD in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. The concentration of enzyme was adjusted to maintain a linear rate of reaction for 1 min to allow accurate rate determination. Reactions were monitored at the optimal wavelength for each substrate using an M200 Infinite Pro plate reader (Tecan). Rates were determined over the initial linear portion of the graph. Quinone reductase assays were performed as described previously [14] with the addition of 1 \u03bcM flavin to the reaction buffer. The effect of FMN concentration on enzymatic activity was measured using Phenol Blue (Phb) as the substrate. The reaction mixture was as follows: 0.2\u201310 \u03bcg/ml azoreductase, 100 \u03bcM Phb, 500 \u03bcM NAD(P)H and 1\u201320 \u03bcM FMN/FAD in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. A fixed concentration of each azore\ufffeductase was used for assays at all concentrations of flavin. Phb reduction was measured via the change in absorbance at 660 nm. Homology modelling A homology model for paMdaB was generated using the phyre2 web server [27] in intensive mode. The paMdaB model was based upon the structures of MdaB from Yersiniae pestis (PDB code 3RPE, 59% identical) and E. coli (PDB code 2AMJ [28], 61% identical)."
  },
  "18337254": {
    "bleu": 92.77171646071145,
    "meteor": 91.14948800181743,
    "rouge1_p": 0.9518796992481203,
    "rouge1_r": 0.9634703196347032,
    "rouge1_f": 0.9576399394856279,
    "rouge2_p": 0.9202407825432656,
    "rouge2_r": 0.9314546839299315,
    "rouge2_f": 0.9258137774413323,
    "rougeL_p": 0.9503759398496241,
    "rougeL_r": 0.9619482496194824,
    "rougeL_f": 0.9561270801815431,
    "cosine_similarity": 0.901285010000149,
    "precision": 0.8130841121495327,
    "recall": 0.8207547169811321,
    "ner_f1": 0.8169014084507042,
    "ner_tp": 87,
    "ner_fp": 20,
    "ner_fn": 19,
    "bertscore_p": 0.9351201057434082,
    "bertscore_r": 0.9333486557006836,
    "bertscore_f1": 0.9344983100891113,
    "bertscore_scibert_error": "The expanded size of the tensor (1950) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1950].  Tensor sizes: [1, 512]",
    "len_gt": 7973,
    "len_jl": 7934,
    "GT": "EXPERIMENTAL PROCEDURES Protein Preparation for Crystallization\u2014 The recombinant AzoR used for crystallization was expressed and purified as described previously (1, 21). Improvement of the Quality of the Tetragonal Crystals of Oxidized AzoR\u2014 The improved oxidized tetragonal crystals (P4\u20842\u20812) were obtained in the same crystallization method as reported previously (21), except that 0.1 mM warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl)coumarin) was added to the crystallization solution as an additive reagent for crystal growth (the electron density of warfarin was not observed). The crystals grew to full size (0.1 \u00d7 0.1 \u00d7 0.6 mm) within 1 week. Chemical Reduction of the Tetragonal Crystals of Oxidized AzoR\u2014 Crystals of the enzyme in the reduced state were prepared by transferring the oxidized tetragonal crystals to a degassed reducing solution containing 200 mM MgCl\u2082, 100 mM HEPES, pH 7.5, 30% (v/v) 2-propanol, 30% (v/v) ethylene glycol, and a saturated concentration of sodium dithionite at 4 \u00b0C. After 10 min, the crystals were transferred to fresh reducing solution. This procedure was repeated two more times. The crystals lost their bright yellow color and became transparent over the course of the treatment. They were then immediately flash-cooled in a stream of nitrogen and stored in liquid nitrogen prior to data collection. It was verified that the crystal remained transparent after data collection. Co-crystallization with Dicoumarol\u2014 The purified protein was dialyzed against a solution containing 10 mM Tris-HCl, pH 8.0, 0.1 mM FMN, 4% (v/v) pyridine, and 2 mM dicoumarol. Crystals of AzoR in complex with dicoumarol were obtained from a drop made by mixing 16 mg/ml of the protein solution mentioned above and an equal volume of reservoir solution containing 200 mM NaCl, 100 mM HEPES, pH 7.5, and 20% (w/v) polyethylene glycol 3000. The drop was equilibrated over the reservoir solution by the hanging-drop vapor diffusion method at 20 \u00b0C. The crystals grew to full size (0.05 \u00d7 0.5 \u00d7 0.5 mm) within 1 week. These crystals belonged to the tetragonal space group P4\u208212. Crystallization of the Orthorhombic Crystals of Oxidized AzoR\u2014 The purified protein was dialyzed against a solution containing 10 mM Tris-HCl, pH 8.0, and 0.1 mM FMN. The orthorhombic (P2\u20812\u20812\u2081) crystals of oxidized AzoR were obtained from a drop made by equal volumes of three solutions: 8 mg/ml of the protein solution mentioned above, 100 mM NAD solution (the electron density of NAD was not observed), and a reservoir solution containing 200 mM NaOAc, 200 mM sodium cacodylate, pH 6.7, 15% (w/v) polyethylene glycol 8000, and 3% (v/v) dimethyl sulfoxide. The drop was equilibrated over the reservoir solution by the hanging-drop vapor diffusion method at 25 \u00b0C. The crystals grew to full size (0.03 \u00d7 0.05 \u00d7 0.5 mm) within 2 weeks. Data Collection and Processing\u2014 All diffraction data were collected under cryogenic conditions at 100 K. Prior to data collection, the crystals were soaked in a reservoir solution containing 30% (v/v) ethylene glycol or 25% (v/v) glycerol as a cryoprotectant. All diffraction data were collected at KEK (Tsukuba, Japan). The beamlines used are shown in Table 1. Data were reduced with MOSFLM, SCALA, and TRUNCATE from the CCP4 program suite (22). Structure Determination\u2014 The initial structures of all the crystals were obtained by molecular replacement with MOLREP (23) using the 1.8 \u00c5 resolution structure of oxidized AzoR as a search model (8). The solutions were then improved by ARP/wARP (24) followed by iterative manual model building with XtalView (25). All the stages of maximum likelihood refinement were carried out with REFMAC5 (26). For the refinement of the oxidized and reduced tetragonal crystal structures, the restraint of the planarity of the isoalloxazine ring was removed from the standard REFMAC5 library to allow the model to adopt the omit density map more precisely. Model Analysis\u2014 The quality of the model was checked with PROCHECK (27). LSQKAB was used to superpose the molecules and to calculate the root mean square deviation between pairs of equivalent C\u03b1 atoms and all atoms of the proteins (28). Structure figures were prepared with PyMOL (29). Mutant Preparations\u2014 For enzymatic analyses, mutations and C-terminal His tag were introduced into the AzoR gene by two rounds of PCR with pETacpD as a template (1). The NADH-methyl red reductase activity of the His-tagged wild-type AzoR was very similar to that of the non-tagged wild-type AzoR. To mutate Arg-59 to Ala, the following pairs of oligonucleotide primers were used for the first PCR: 5\u2032-terminal sense primer for AzoR (5\u2032-GGGAATTCCATATGAGCAAGGTATTAGTTCTTAAATCCAGC-3\u2032 containing an NdeI site) and R59A mutation antisense primer (5\u2032-CGGCGCATCGCTCGGAGCCAGAGCGCCAACCAGTTC-3\u2032) were used to amplify the 5\u2032-terminal part of the AzoR gene. R59A mutation sense primer (5\u2032-GAACTGGTTGGCGCTCTGGCTCCGAGCGATGCGCCG-3\u2032) and 3\u2032-terminal antisense primer for AzoR (5\u2032-AAACCGCTCGAGTTAGTGATGGTGATGGTGGTGTGCAGAAACAATGCTGTCGATGGC-3\u2032 containing an XhoI site and His\u2086 tag sequence) were used to amplify the 3\u2032-terminal part of the AzoR gene. PCR products were used as templates for the second PCR, which used the 5\u2032-terminal sense primer and 3\u2032-terminal antisense primer for AzoR. To generate other mutants, the following primers were used in place of R59A mutation sense and antisense primers: Y120A mutation sense primer (5\u2032-GCAGGCGTTACTTTCCGCGCTACCGAGAACGGTCCG-3\u2032) and Y120A mutation antisense primer (5\u2032-CGGACCGTTCTCGGTAGCGCGGAAAGTAACGCCTGC-3\u2032) for Tyr-120 to Ala mutant; F162A mutation sense primer (5\u2032-CCACGTTCCTCGGCGCTATCGGCATTACCGATG-3\u2032) and F162A mutation antisense primer (5\u2032-CATCGGTAATGCCGATAGCGCCGAGGAACGTGG-3\u2032) for Phe-162 to Ala mutant. To generate the His-tagged wild-type AzoR gene as a control that is catalytically very similar to AzoR, the 5\u2032-terminal sense primer and the 3\u2032-terminal antisense primer for AzoR described above were used for PCR with pETacpD as a template. The PCR product of each mutated AzoR was inserted between the NdeI and the XhoI sites of the expression vector pET-22b (Novagen). The entire DNA sequence was confirmed by DNA sequencing. His-tagged wild-type and mutant AzoR proteins were expressed in E. coli BL21(DE3) at 37 \u00b0C in Luria Bertani medium containing 100 \u00b5g/ml ampicillin. Protein expression was induced by adding 1 mM isopropyl-\u03b2-D-thiogalactopyranoside to early exponential phase cultures (A600 \u2248 0.5) for 3 h. Bacteria were lysed in a solution containing 50 mM Tris-HCl, pH 7.5, 2 mM 2-mercaptoethanol, 500 mM NaCl, 20 mM imidazole, and 10 mg/ml lysozyme by sonication. The lysate was centrifuged, and the His-tagged proteins were purified by gravity-flow chromatography using nickel-nitrilotriacetic acid-agarose (Qiagen) according to the manufacturer\u2019s instructions. Eluted proteins were stored at \u221280 \u00b0C in a solution containing 20 mM Tris-HCl, pH 7.5, 2 mM 2-mercaptoethanol, 200 mM NaCl, and 0.1 mM FMN. Protein samples were analyzed on SDS-PAGE and were more than 95% pure. Enzyme Assays\u2014 The NADH-methyl red reductase activity for each mutant was determined spectrophotometrically by a method described previously (1). The initial reaction rates were fitted to the Equation 1 1 \ud835\udc63 = 1 \ud835\udc49 + \ud835\udc3e \ud835\udc34 \ud835\udc49 \ud835\udc34 + \ud835\udc3e \ud835\udc35 \ud835\udc49 \ud835\udc35 v 1 \u200b = V 1 \u200b + VA K A \u200b \u200b + VB K B \u200b \u200b where v is the initial reaction rate, V is the maximum reaction rate at infinite substrate concentrations, A and B are the concentrations of methyl red and NADH, respectively, and K\u2090 and K_b are their corresponding Michaelis constants. The initial reaction rate of p-methyl red reduction was determined in the same manner as methyl red, except that a 460-nm wavelength was used to monitor the decrease in absorbance of p-methyl red and a molar absorption coefficient of 17,310 M\u207b\u00b9 cm\u207b\u00b9 was used. K_b was applied to obtain Michaelis constants for p-methyl red because K_b does not depend on the kind of azo compound. All of these assays were performed in triplicate.",
    "LLM": "EXPERIMENTAL PROCEDURES Protein Preparation for Crystallization\u2014The recombinant AzoR used for crystallization was expressed and purified as described previously (1, 21). Improvement of the Quality of the Tetragonal Crystals of Oxi\ufffedized AzoR\u2014The improved oxidized tetragonal crystals (P42212) were obtained in the same crystallization method as reported previously (21), except that 0.1 mM warfarin (4-hy\ufffedroxy-3-(3-oxo-1-phenylbutyl)coumarin) was added to the crystallization solution as an additive reagent for crystal growth (the electron density of warfarin was not observed). The crystals grew to full size (0.1 0.1 0.6 mm) within 1 week. Chemical Reduction of the Tetragonal Crystals of Oxidized AzoR\u2014Crystals of the enzyme in the reduced state were pre\ufffepared by transferring the oxidized tetragonal crystals to a degassed reducing solution containing 200 mM MgCl2, 100 mM HEPES, pH 7.5, 30% (v/v) 2-propanol, 30% (v/v) ethylene glycol, and a saturated concentration of sodium dithionite at 4 \u00b0C. After 10 min, the crystals were transferred to fresh reducing solution. This procedure was repeated two more times. The crystals lost their bright yellow color and became transparent over the course of the treatment. They were then immediately flash-cooled in a stream of nitrogen and stored in liquid nitro\ufffegen prior to data collection. It was verified that the crystal remained transparent after data collection. Co-crystallization with Dicoumarol\u2014The purified protein was dialyzed against a solution containing 10 mM Tris-HCl, pH 8.0, 0.1 mM FMN, 4% (v/v) pyridine, and 2 mM dicoumarol. Crystals of AzoR in complex with dicoumarol were obtained from a drop made by mixing 16 mg/ml of the protein solution mentioned above and an equal volume of reservoir solution containing 200 mM NaCl, 100 mM HEPES, pH 7.5, and 20% (w/v) polyethylene glycol 3000. The drop was equilibrated over the reservoir solution by the hanging-drop vapor diffusion method at 20 \u00b0C. The crystals grew to full size (0.05 0.5 0.5 mm) within 1 week. These crystals belonged to the tetragonal space group P4212. Crystallization of the Orthorhombic Crystals of Oxidized AzoR\u2014The purified protein was dialyzed against a solution containing 10 mM Tris-HCl, pH 8.0, and 0.1 mM FMN. The orthorhombic (P212121) crystals of oxidized AzoR were obtained from a drop made by equal volumes of three solutions: 8 mg/ml of the protein solution mentioned above, 100 mM NAD solution (the electron density of NAD was not observed), and a reservoir solution containing 200 mM NaOAc, 200 mM sodium cacodylate, pH 6.7, 15% (w/v) polyethylene glycol 8000, and 3% (v/v) dimethyl sulfoxide. The drop was equilibrated over the reservoir solution by the hanging-drop vapor diffusion method at 25 \u00b0C. The crystals grew to full size (0.03 0.05 0.5 mm) within 2 weeks. Data Collection and Processing\u2014All diffraction data were collected under cryogenic conditions at 100 K. Prior to data collection, the crystals were soaked in a reservoir solution con\ufffetaining 30% (v/v) ethylene glycol or 25% (v/v) glycerol as a cryo\ufffeprotectant. All diffraction data were collected at KEK (Tsukuba, Japan). The beamlines used are shown in Table 1. Data were reduced with MOSFLM, SCALA, and TRUNCATE from the CCP4 program suite (22). Structure Determination\u2014The initial structures of all the crystals were obtained by molecular replacement with MOL\ufffeREP (23) using the 1.8 \u00c5 resolution structure of oxidized AzoR as a search model (8). The solutions were then improved by ARP/wARP (24) followed by iterative manual model building with XtalView (25). All the stages of maximum likelihood refinement were carried out with REFMAC5 (26). For the refinement of the oxidized and reduced tetragonal crystal structures, the restraint of the planarity of the isoalloxazine ring was removed from the standard REFMAC5 library to allow the model to adopt the omit density map more precisely. Model Analysis\u2014The quality of the model was checked with PROCHECK (27). LSQKAB was used to superpose the mole\ufffecules and to calculate the root mean square deviation between pairs of equivalent C\u0001 atoms and all atoms of the proteins (28). Structure figures were prepared with PyMOL (29). Mutant Preparations\u2014For enzymatic analyses, mutations and C-terminal His tag were introduced into the AzoR gene by two rounds of PCR with pETacpD as a template (1). The NADH-methyl red reductase activity of the His-tagged wild\ufffetype AzoR was very similar to that of the non-tagged wild-type AzoR. To mutate Arg-59 to Ala, the following pairs of oligonucleo\ufffetide primers were used for the first PCR: 5\u0001-terminal sense primer for AzoR (5\u0001-GGGAATTCCATATGAGCAAGGTAT\ufffeTAGTTCTTAAATCCAGC-3\u0001 containing an NdeI site) and R59A mutation antisense primer (5\u0001-CGGCGCATCGCTCG\ufffeGAGCCAGAGCGCCAACCAGTTC-3\u0001) were used to amplify the 5\u0001-terminal part of the AzoR gene. R59A mutation sense primer (5\u0001-GAACTGGTTGGCGCTCTGGCTCCGAGCGA\ufffeTGCGCCG-3\u0001) and 3\u0001-terminal antisense primer for AzoR (5\u0001- AAACCGCTCGAGTTAGTGATGGTGATGGTGGTGTG\ufffeCAGAAACAATGCTGTCGATGGC-3\u0001 containing an XhoI site and His6 tag sequence) were used to amplify the 3\u0001-ter\ufffeminal part of the AzoR gene. PCR products were used as templates for the second PCR, which used the 5\u0001-terminal sense primer and 3\u0001-terminal antisense primer for AzoR. To generate other mutants, the following primers were used in place of R59A mutation sense and antisense primers: Y120A mutation sense primer (5\u0001-GCAGGCGTTACTTTCCGCG\ufffeCTACCGAGAACGGTCCG-3\u0001) and Y120A mutation anti\ufffesense primer (5\u0001-CGGACCGTTCTCGGTAGCGCGGAA\ufffeAGTAACGCCTGC-3\u0001) for Tyr-120 to Ala mutant; F162A mutation sense primer (5\u0001-CCACGTTCCTCGGCGCTATC\ufffeGGCATTACCGATG-3\u0001) and F162A mutation antisense primer (5\u0001-CATCGGTAATGCCGATAGCGCCGAGGAAC\ufffeGTGG-3\u0001) for Phe-162 to Ala mutant. To generate the His\ufffetagged wild-type AzoR gene as a control that is catalytically very similar to AzoR, the 5\u0001-terminal sense primer and the 3\u0001-termi\ufffenal antisense primer for AzoR described above were used for PCR with pETacpD as a template. The PCR product of each mutated AzoR was inserted between the NdeI and the XhoI sites of the expression vector pET-22b (Novagen). The entire DNA sequence was confirmed by DNA sequencing. His-tagged wild-type and mutant AzoR proteins were expressed in E. coli BL21(DE3) at 37 \u00b0C in Luria Bertani medium containing 100 g/ml ampicillin. Protein expression was induced by adding 1 mM isopropyl--D-thiogalactopyran\ufffeoside to early exponential phase cultures (A600 \u0004 0.5) for 3 h. Bacteria were lysed in a solution containing 50 mM Tris-HCl, pH 7.5, 2 mM 2-mercaptoethanol, 500 mM NaCl, 20 mM imid\ufffeazole, and 10 mg/ml lysozyme by sonication. The lysate was centrifuged, and the His-tagged proteins were purified by grav\ufffeity-flow chromatography using nickel-nitrilotriacetic acid-aga\uffferose (Qiagen) according to the manufacturer\u2019s instructions. Eluted proteins were stored at \u000580 \u00b0C in a solution containing 20 mM Tris-HCl, pH 7.5, 2 mM 2-mercaptoethanol, 200 mM NaCl, and 0.1 mM FMN. Protein samples were analyzed on SDS-PAGE and were more than 95% pure. Enzyme Assays\u2014The NADH-methyl red reductase activity for each mutant was determined spectrophotometrically by a method described previously (1). The initial reaction rates were fitted to the Equation 1 1 v \u0004 1 V \u0005 KA VA \u0005 KB VB (Eq. 1) where v is the initial reaction rate, V is the maximum reaction rate at infinite substrate concentrations, A and B are the con\ufffecentrations of methyl red and NADH, respectively, and KA and KB are their corresponding Michaelis constants. The initial reaction rate of p-methyl red reduction was determined in the same manner as methyl red, except that a 460-nm wavelength was used to monitor the decrease in absorbance of p-methyl red and a molar absorption coefficient of 17310 M\u00051 cm\u00051 was used.KB was applied to obtain Michaelis constants for p-methyl red because KB does not depend on the kind of azo compound. All of these assays were performed in triplicate."
  },
  "30342139": {
    "bleu": 97.11891042430591,
    "meteor": 97.38185547038736,
    "rouge1_p": 0.9610169491525423,
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    "rouge1_f": 0.9681274900398407,
    "rouge2_p": 0.9366873940079141,
    "rouge2_r": 0.9506597819850832,
    "rouge2_f": 0.943621867881549,
    "rougeL_p": 0.96045197740113,
    "rougeL_r": 0.9747706422018348,
    "rougeL_f": 0.9675583380762662,
    "cosine_similarity": 0.92718149066618,
    "precision": 0.9183673469387755,
    "recall": 0.9507042253521126,
    "ner_f1": 0.9342560553633218,
    "ner_tp": 135,
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    "bertscore_p": 0.9706100821495056,
    "bertscore_r": 0.9802026748657227,
    "bertscore_f1": 0.9754934310913086,
    "bertscore_scibert_error": "The expanded size of the tensor (2396) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2396].  Tensor sizes: [1, 512]",
    "len_gt": 9975,
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    "GT": "2. Materials and methods 2.1. Organism growth condition Azo dye degrading bacteria C. violaceum (MTCC 2656) was commercially purchased from Microbial Type Culture Collection (MTCC) India. The C. violaceum was grown in media containing Beef extract, yeast extract, peptone and NaCl or in the nutrient agar plate. Cultured cells ware grown at 37 \u00b0C for overnight in a shaking incubator at the speed of 180 rpm [18]. All the chemicals used during the experiment are purchased from Sigma (USA) and HiMedia (Mumbai, India) in analytical grade. 2.2. Cloning of CV_RS09840 in pET28a(+) expression vector Genomic DNA was isolated from C. violaceum using nucleotide sequence of azoreductase from C. violaceum (CV_RS09840) strain was identified from the NCBI database with accession number NC_005085.1. Primers specific for the azoreductase gene were used for the amplification of full length azoreductase gene. Forward primer CV Azo F: 5\u2032-CGGGATCCATGAAACTGCTCCACCTCG-3\u2032 (underlined sequence is restriction site for BamHI and highlighted sequences are for start codon) and reverse primer CV AzoR: 5\u2032-CCGTCGACTTACAGCGCCAACTTGGC-3\u2032 (underlined sequence is restriction site for SalI and highlighted sequences for stop codon) specific for CV_RS09840 gene (NCBI reference number: NC_005085.1). The BamHI (NEB) and SalI (NEB) restriction site was specifically incorporated in the primers. PCR reaction was performed in 40 \u03bcl reaction volume containing 20 \u03bcl of Dream Taq green PCR master mix (Fermentas), 0.1 \u03bcg of each primer, 0.1 \u03bcg of C. violaceum (CV_RS09840) genomic DNA was mixed and final volume was adjusted by adding nuclease free water. The PCR reaction conditions are: initial denaturation 3 min at 95 \u00b0C; 30 cycle of denaturation for 15 s at 95 \u00b0C; annealing 30 s at 62 \u00b0C; elongation 40 s at 72 \u00b0C and final extension for 10 min at 72 \u00b0C. The PCR reaction amplifies the 609 bp of amplicon shown on a 1% agarose gel stained with Etbr. The PCR product and pET28a (+) (Novagen) was restriction digested with BamHI and SalI, and ligated to the pET28a (+) vector by T4 DNA ligase. The recombinant pET28a (+) were transformed in E. coli DH5\u03b1 cells. All the cells were screened for the presence of CV_RS09840. The initial confirmation was done by PCR amplification of azoreductase gene by using gene specific primers. Clone was further confirmed by restriction digestion with BamHI and SalI showing release of 609 bp and finally the presence of azoreductase (CV_RS09840) was verified by sequencing using T7 forward and reverse primer. 2.3. Expression and purification of recombinant azoreductase Recombinant pET28a-CV_RS09840 (6\u00d7 Histidine tag at the N terminal) was isolated from transformed E. coli DH5\u03b1 cells and further transformed into E. coli BL21 (DE3) cells for soluble expression. The transformed cells of E. coli BL21 (DE3) were grown in 5 ml of LB media as a primary culture at 37 \u00b0C in the shaking incubator at 180 rpm with 50 \u03bcg/\u03bcl of kanamycin (HiMedia) to avoid unnecessary growth. Secondary culture of transformed E. coli BL21 (DE3) cells after reaching an optical density of around 0.4 at A600 was shifted to shaking incubator maintained at 20 \u00b0C, 180 rpm for 8 h. 2.4. Protein purification For protein purification, secondary culture cells was grown for 8 h at 16 \u00b0C and this cultured cells was centrifuged at 5000 rpm for 10 min and sonicated with 6 s on and 10 s off cycle sonicator (Sonics) with lysis buffer (20 mM Tris-HCl, 250 mM NaCl pH 7.5 with 2.5% Glycerol). The sonicated sample was centrifuged at 12,000 rpm for 40 min at 4 \u00b0C. The clarified supernatant was collected and loaded on the pre equilibrated Ni-Nitrilotriacetic acid column (with 10 mM imidazole in 20 mM Tris-HCl and 250 mM NaCl pH 7.5) for purification and protein was eluted with elution buffer (250 mM imidazole in 20 mM Tris-HCl and 250 mM NaCl). The purified protein was dialyzed against potassium phosphate buffer (25 mM KH\u2082PO\u2084 and 25 mM K\u2082HPO\u2084 pH 7.2) overnight for the removal of residual imidazole. The size of purified azoreductase enzyme was estimated by running SDS-PAGE (12%). Concentration of purified protein was estimated by Bradford assay using the Bovine serum albumin (BSA) as a standard [19]. 2.5. Kinetic characterization of enzymes Enzyme activity of purified recombinant azoreductase was measured by decreasing the concentration of NADH using method reported earlier [20]. The azo bonds present in the dye are reduced when reaction starts proceeding with the addition of enzyme. The initial velocity of reaction was monitored by changing the amount of substrate in a glass cuvette of 1.0 cm path length and absorbance was taken at 340 nm. The optical density was measured in Agilent Technology Carry 100 UV\u2013Vis spectrophotometer at room temperature. The standard reaction volume was 1 ml which contained 25 mM K\u2082HPO\u2084, 25 mM KH\u2082PO\u2084 pH 7.2 (HiMedia) varying concentration (1 nM\u20131000 nM) of Methyl Red, Methyl Orange or amaranth azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of enzyme. The reaction mixture was incubated in a water bath at 30 \u00b0C for 1 min before taking the absorbance and the data obtained was fitted in Michaelis-Menten equation and double reciprocal plot through which Km and Vmax were estimated. 2.6. pH optimum The pH optimum studies were carried out at a pH range of 4.0\u201310.0. The reaction mixture contains two buffers: 25 mM K\u2082HPO\u2084 (pH 4.0\u201311.0), 25 mM KH\u2082PO\u2084 (pH 4.0\u201311.0), 1 \u03bcM of azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of enzyme. The reaction was initiated after the addition of enzyme followed by incubation in water bath at 30 \u00b0C and absorbance was taken at 340 nm. The pH optimum was determined by plotting the relative activity at different pH values. 2.7. Temperature optimum The temperature optimum studies were carried out at different temperatures ranging from 10 \u00b0C to 60 \u00b0C. The reaction mixture contains two buffers: 25 mM K\u2082PO\u2084 (pH 7.2), 25 mM KH\u2082PO\u2084 (pH 7.2), 1 \u03bcM of azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of enzyme. The reaction was initiated after the addition of enzyme followed by incubation in water bath at different temperature range from 10 \u00b0C to 60 \u00b0C and absorbance was taken 340 nm. Appropriate substrate blanks were included in all the reactions. The temperature optimum was determined by plotting the relative activity at different temperature values. 2.8. Prosthetic group identification of enzyme The prosthetic group present within the enzyme flavin adenine dinucleotide (FAD) was confirmed by HPLC using earlier reported method after minor modification [14,21,22]. High purity and analytical grade FAD purchased from Sigma Aldrich was used as a standard. In brief, 0.5 ml of recombinant azoreductase (0.4 \u03bcg \u03bcl\u207b\u00b9) present in 25 mM phosphate buffer (pH 7.2) was taken and denatured in water bath at 80 \u00b0C for 10 min and centrifuged at 10,000 rpm for 10 min to remove the denatured protein. Supernatant was collected and used after filtering with 0.4 mm filter to measure the presence of FAD in Thermo Scientific HPLC ultimate 3000 system connected with UV\u2013Vis detector and a reverse-phase C18 column (5 \u03bcm 120 \u00c5 4.5 \u00d7 259 mm). The mobile phase was 25 mM phosphate buffer (pH 7.2) and acetonitrile (4:8, v/v) with a flow rate of 0.5 ml min\u207b\u00b9. The presence of FAD was monitored at 260 nm. The standard FAD was also dissolved in 25 mM phosphate buffer and used as control. 2.9. Degradation kinetics study of dyes The biodegradation product of methyl red, methyl orange and amaranth dyes was monitored by using the UV\u2013Visible spectrophotometer (Agilent Technology Carry-100) and Fourier Transform Infrared Spectroscopy (Perkin Elmer Spectrum two) analysis. The reaction for UV\u2013Vis spectroscopy contain 25 mM potassium phosphate buffer, 1 \u03bcM of azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of azoreductase enzyme. The reaction starts by adding the azoreductase enzyme in the sample and scanning the consumption of NADH used in this reaction at 340 nm. The biodegraded product was analysed after lyophilisation (Labconco) in Fourier Transform Infrared Spectroscopy (Perkin Elmer Spectrum two) and compared with control dye. The FTIR analysis was done in Mid of Infrared region from 400 to 4000 cm\u207b\u00b9 with 20 scan speed. 2.10. Toxicity assay The toxicity of methyl red, methyl orange and amaranth dye was checked on fibroblast cell line (L929), to find out the probable health impacts. In toxicity assay, the fibroblast cell line (L929) was procured from National Centre for Cell Sciences (NCCS) Pune India, and maintained in RPMI 1640 liquid media supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 U penicillin and 100 \u03bcg ml\u207b\u00b9 streptomycin was used for assessing the toxicity assay with Methyl red, methyl orange and amaranth dye. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay was used to check the toxicity efficacy of the dyes. Healthy cells (5000) seeded in the 96 well plates and allowed them to adhere for 24 h at 37 \u00b0C. After 24 h cells were treated with varying concentration (20 \u03bcg to 1000 \u03bcg) of dye and incubated again at 37 \u00b0C for 24 h. After completion of 24 h media was removed from each well and 200 \u03bcl of MTT (0.5 mg ml\u207b\u00b9) was added in to them and again incubated for 4 h at 37 \u00b0C, after completion of 4 h all the MTT was discarded and 100 \u03bcl DMSO was added to each plate and mixed properly. Absorbance was taken at 570 nm in micro plate reader by (BIOTEK Synergy HT) [23]. 2.11. Stability of azoreductase with various denaturant The ability of purified enzyme to retain its activity under denaturant conditions and organic solvents, 2 \u03bcg of enzyme was incubated with different concentration of Urea, GdmCl and SDS for 12 h at room temperature and activity was measured in spectrophotometer. Stability of azoreductase was measured with respect to percent activity of enzyme. The reaction mixture contains amaranth dye, FAD, NADH and Enzyme. The reaction was started by adding enzyme that was already incubated with different denaturant (Urea, GdmCl and SDS) for 12 h.",
    "LLM": "2. Materials and methods 2.1. Organism growth condition Azo dye degrading bacteria C. violaceum (MTCC 2656) was commer\ufffecially purchased from Microbial Type Culture Collection (MTCC) India. The C. violaceum was grown in media containing Beef extract, yeast ex\ufffetract, peptone and NaCl or in the nutrient agar plate. Cultured cells ware grown at 37 \u00b0C for overnight in a shaking incubator at the speed of 180 rpm [18]. All the chemicals used during the experiment are pur\ufffechased from Sigma (USA) and HiMedia (Mumbai, India) in analytical grade. 2.2. Cloning of CV_RS09840 in pET28a(+) expression vector Genomic DNA was isolated from C. violaceum using nucleotide se\ufffequence of azoreductase from C. violaceum (CV_RS09840) strain was identified from the NCBI database with accession number NC_005085. 1. Primers specific for the azoreductase gene were used for the amplifi\ufffecation of full length azoreductase gene. Forward primer CV Azo F: 5\u2032- CGGGATCCATGAAACTGCTCCACCTCG-3\u2032 (underlined sequence is re\ufffestriction site for BamHI and highlighted sequences are for start codon) and reverse primer CV AzoR: 5\u2032-CCGTCGACTTACAGCGCCAACTTGGC-3\u2032 (underlined sequence is restriction site for SalI and highlighted se\ufffequences for stop codon) specific for CV_RS09840 gene (NCBI reference number: NC_005085.1). The BamHI (NEB) and SalI (NEB) restriction site was specifically incorporated in the primers. PCR reaction was per\ufffeformed in 40 \u03bcl reaction volume containing 20 \u03bcl of Dream Taq green PCR master mix (Fermentas), 0.1 \u03bcg of each primer, 0.1 \u03bcg of C. violaceum (CV_RS09840) genomic DNA was mixed and final volume was adjusted by adding nuclease free water. The PCR reaction condi\ufffetions are: initial denaturation 3 min at 95 \u00b0C; 30 cycle of denaturation for 15 s at 95 \u00b0C; annealing 30 s at 62 \u00b0C; elongation 40 s at 72 \u00b0C and final extension for 10 min at 72 \u00b0C. The PCR reaction amplifies the 609 bp of amplicon shown on a 1% agarose gel stained with Etbr. The PCR product and pET28a (+) (Novagen) was restriction digested with BamHI and SalI, and ligated to the pET28a (+) vector by T4 DNA ligase. The recombinant pET28a (+) were transformed in E. coli DH5\u03b1 cells. All the cells were screened for the presence of CV_RS09840. The initial con\ufffefirmation was done by PCR amplification of azoreductase gene by using gene specific primers. Clone was further confirmed by restriction diges\ufffetion with BamHI and SalI showing release of 609 bp and finally the pres\ufffeence of azoreductase (CV_RS09840) was verified by sequencing using T7 forward and reverse primer. 2.3. Expression and purification of recombinant azoreductase Recombinant pET28a-CV_RS09840 (6\u00d7 Histidine tag at the N termi\ufffenal) was isolated from transformed E. coli DH5\u03b1 cells and further trans\ufffeformed into E. coli BL21 (DE3) cells for soluble expression. The transformed cells of E. coli BL21 (DE3) were grown in 5 ml of LB media as a primary culture at 37 \u00b0C in the shaking incubator at 180 rpm with 50 \u03bcg/\u03bcl of kanamycin (HiMedia) to avoid unnecessary growth. Secondary culture of transformed E. coli BL21 (DE3) cells after reaching an optical density of around 0.4 at A600 was shifted to shaking incubator maintained at 20 \u00b0C, 180 rpm for 8 h. 2.4. Protein purification For protein purification, secondary culture cells was grown for 8 h at 16 \u00b0C and this cultured cells was centrifuged at 5000 rpm for 10 min and sonicated with 6 s on and 10 s off cycle sonicator (Sonics) with lysis buffer (20 mM Tris-HCl, 250 mM NaCl pH 7.5 with 2.5% Glycerol). The sonicated sample was centrifuged at 12,000 rpm for 40 min at 4 \u00b0C. The clarified supernatant was collected and loaded on the pre equili\ufffebrated Ni-Nitrilotriacetic acid column (with 10 mM imidazole in 20 mM Tris-HCl and 250 mM NaCl pH 7.5) for purification and protein was eluted with elution buffer (250 mM imidazole in 20 mM Tris-HCl and 250 mM NaCl). The purified protein was dialyzed against potassium phosphate buffer (25 mM KH2PO4 and 25 mM K2HPO4 pH 7.2) over\ufffenight for the removal of residual imidazole. The size of purified azoreductase enzyme was estimated by running SDS-PAGE (12%). Con\ufffecentration of purified protein was estimated by Bradford assay using the Bovine serum albumin (BSA) as a standard [19]. 2.5. Kinetic characterization of enzymes Enzyme activity of purified recombinant azoreductase was mea\ufffesured by decreasing the concentration of NADH using method reported earlier [20]. The azo bonds present in the dye are reduced when reaction starts proceeding with the addition of enzyme. The initial velocity of re\ufffeaction was monitored by changing the amount of substrate in a glass cu\ufffevette of 1.0 cm path length and absorbance was taken at 340 nm. The optical density was measured in Agilent Technology Carry 100 UV\u2013Vis spectrophotometer at room temperature. The standard reaction volume was 1 ml which contained 25 mM K2HPO4, 25 mM KH2PO4 pH 7.2 (HiMedia) varying concentration (1 nM\u20131000 nM) of Methyl Red, Methyl Orange or amaranth azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of enzyme. The reaction mixture was incubated in a water bath at 30 \u00b0C for 1 min before taking the absorbance and the data obtained was fitted in Michaelis-Menten equation and double reciprocal plot through which Km and Vmax were estimated. 2.6. pH optimum The pH optimum studies were carried out at a pH range of 4.0\u201310.0. The reaction mixture contains two buffers: 25 mM K2HPO4 (pH 4.0\u201311.0), 25 mM KH2PO4 (pH 4.0\u201311.0), 1 \u03bcM of azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of enzyme. The reaction was initi\ufffeated after the addition of enzyme followed by incubation in water bath at 30 \u00b0C and absorbance was taken at 340 nm. The pH optimum was de\ufffetermined by plotting the relative activity at different pH values. 2.7. Temperature optimum The temperature optimum studies were carried out at different tem\ufffeperatures ranging from 10 \u00b0C to 60 \u00b0C. The reaction mixture contains two buffers: 25 mM K2PO4 (pH 7.2), 25 mM KH2PO4 (pH 7.2), 1 \u03bcM of azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of enzyme. The reaction was initiated after the addition of enzyme followed by incubation in water bath at different temperature range from 10 \u00b0C to 60 \u00b0C and absor\ufffebance was taken 340 nm. Appropriate substrate blanks were included in all the reactions. The temperature optimum was determined by plotting the relative activity at different temperature values. 2.8. Prosthetic group identification of enzyme The prosthetic group present within the enzyme flavin adenine di\ufffenucleotide (FAD) was confirmed by HPLC using earlier reported method after minor modification [14,21,22]. High purity and analytical grade FAD purchased from Sigma Aldrich was used as a standard. In brief, 0.5 ml of recombinant azoreductase (0.4 \u03bcg \u03bcl \u22121 ) present in 25 mM phosphate buffer (pH 7.2) was taken and denatured in water bath at 80 \u00b0C for 10 min and centrifuged at 10,000 rpm for 10 min to remove the denatured protein. Supernatant was collected and used after filtering with 0.4 mm filter to measure the presence of FAD in Thermo Scientific HPLC ultimate 3000 system connected with UV\u2013Vis detector and a reverse-phase C18 column (5 \u03bcm 120 \u00c5 4.5 \u00d7 259 mm). The mobile phase was 25 mM phosphate buffer (pH 7.2) and acetonitrile (4:8, v/ v) with a flow rate of 0.5 ml min\u22121 . The presence of FAD was monitored at 260 nm. The standard FAD was also dissolved in 25 mM phosphate buffer and used as control. 2.9. Degradation kinetics study of dyes The biodegradation product of methyl red, methyl orange and ama\uffferanth dyes was monitored by using the UV\u2013Visible spectrophotometer (Agilent Technology Carry-100) and Fourier Transform Infrared Spec\ufffetroscopy (Perkin Elmer Spectrum two) analysis. The reaction for UV\u2013 Vis spectroscopy contain 25 mM potassium phosphate buffer, 1 \u03bcM of azo dye, 0.1 mM NADH, 10 \u03bcM FMN and 2 \u03bcg of azoreductase enzyme. The reaction starts by adding the azoreductase enzyme in the sample and scanning the consumption of NADH used in this reaction at 340 nm. The biodegraded product was analysed after lyophilisation (Labconco) in Fourier Transform Infrared Spectroscopy (Perkin Elmer Spectrum two) and compared with control dye. The FTIR analysis was done in Mid of Infrared region from 400 to 4000 cm\u22121 with 20 scan speed. 2.10. Toxicity assay The toxicity of methyl red, methyl orange and amaranth dye was checked on fibroblast cell line (L929), to find out the probable health impacts. In toxicity assay, the fibroblast cell line (L929) was procured from National Centre for Cell Sciences (NCCS) Pune India, and maintained in RPMI 1640 liquid media supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 U penicillin and 100 \u03bcg ml\u22121 streptomycin was used for assessing the toxicity assay with Methyl red, methyl orange and amaranth dye. MTT [3\u2011(4,5\u2011dimethylthiazol\u20112\u2011yl)\u20112,5\u2011diphenyl\u2011tetrazolium bromide] assay was used to check the toxicity efficacy of the dyes. Healthy cells (5000) seeded in the 96 well plates and allowed them to adhere for 24 h at 37 \u00b0C. After 24 h cells were treated with varying concentration (20 \u03bcg to 1000 \u03bcg) of dye and incubated again at 37 \u00b0C for 24 h. After completion of 24 h media was removed from each well and 200 \u03bcl of MTT (0.5 mg ml\u22121 ) was added in to them and again incubated for 4 h at 37 \u00b0C, after completion of 4 h all the MTT was discarded and 100 \u03bcl DMSO was added to each plate and mixed properly. Absorbance was taken at 570 nm in micro plate reader by (BIOTEK Synergy HT) [23]. 2.11. Stability of azoreductase with various denaturant The ability of purified enzyme to retain its activity under denaturant conditions and organic solvents, 2 \u03bcg of enzyme was incubated with dif\ufffeferent concentration of Urea, GdmCl and SDS for 12 h at room tempera\ufffeture and activity was measured in spectrophotometer. Stability of azoreductase was measured with respect to percent activity of enzyme. The reaction mixture contains amaranth dye, FAD, NADH and Enzyme. The reaction was started by adding enzyme that was already incubated with different denaturant (Urea, GdmCl and SDS) for 12 h."
  },
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    "bertscore_p": 0.9870052337646484,
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    "bertscore_scibert_error": "The expanded size of the tensor (3522) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 3522].  Tensor sizes: [1, 512]",
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    "GT": "MATERIALS AND METHODS Screening of dye-decolorizing microorganisms. Three Bacillus sp. isolates (Bacillus sp. strain SF, Bacillus sp. strain LF, and Bacillus pallidus), previously isolated from a wastewater drain of a textile finishing company (27), were incubated on petri dishes for 1 week at 50\u00b0C with the following medium (sterile): 250 ml of tap water, 9.25 g of standard I nutrient agar (Merck KGaA), 1.05 g of NaHCO3, and 10 mg of dye. The pH was controlled to 7.0 and 9.5, and growth and decolorization were monitored visually. Further experiments were carried out in 300-ml baffled Erlenmeyer flasks on a rotary shaker at 65\u00b0C and 160 rpm. The cultivation medium consisted of the following (in grams per liter): yeast extract (Merck), 3.0; peptone from casein (Merck), 3.0; NaHCO3, 4.2; KH2PO4, 1.0; and 1 ml of trace element solution SL-6 (29) per liter. The pH was adjusted to 9.0 and the dye concentration was 0.1 g liter\u20131. Flasks containing 90 ml of medium were inoculated with 10 ml of microorganism suspension from precultures, and dyes were added from stock solutions. The dyes Mordant Black 9 and Mordant Black 96 were from Dyestar (Frankfurt, Germany), Acid Blue 225 and Disperse Red 86 were from Ciba (Basel, Switzerland), and all other dyes were from Sigma. All dye names are given according to the color index (http://www.colour-index.org ) when structures are available. Growth was monitored under a microscope, and dye degradation was measured by spectrophotometric determination of the absorbance at the wavelength maximum for each dye. Blanks were run by adding 100 mg of thiomersal [sodium ethyl-(2-mercaptobenzoato-(2)-O,S)-mercurate] per liter to prevent growth, taking into consideration the possible adsorption of the dyestuff on biomass. Cultivation of Bacillus sp. strain SF. Bacillus strain SF was grown in 300-ml baffled Erlenmeyer flasks on a rotary shaker at 60\u00b0C and 160 rpm in a medium consisting of the following components (in grams per liter): KH2PO4, 3.5; Na2HPO4 \u00b7 7H2O, 7.5; yeast extract (Merck), 10.0; peptone from casein (Merck), 20.0; (NH4)2SO4, 2.5; MgSO4 \u00b7 7H2O, 4.5; MnSO4 \u00b7 H2O, 0.2; iron citrate \u00b7 5H2O, 0.7; and 2.5% (vol/vol) SL-6 trace element solution, as described previously (29). SL-6 trace element solution contained the following (in milligrams per liter): ZnSO4 \u00b7 7H2O, 100.0; MnCl2 \u00b7 4H2O, 30.0; H3BO3, 300.0; CuCl2 \u00b7 2H2O, 10.0; NiCl2 \u00b7 6H2O, 20.0; Na2MoO4 \u00b7 2H2O, 30.0; and CoCl2 \u00b7 6H2O, 200.0. To avoid precipitation of salts during sterilization in an autoclave, MnSO4 \u00b7 H2O, MgSO4 \u00b7 7H2O, and iron citrate \u00b7 5H2O were autoclaved separately, and the two solutions were combined sterilely after they were cooled in a laminar flow hood. Ten-liter fermentations were carried out in a Chemap-CF 2000 fermentor, using the cultivation medium described above. The fermentor was equipped with pH and oxygen controls. Precultures were grown in baffled Erlenmeyer flasks as described above. Downstream processing. Cells were harvested at the end of the exponential phase of growth and centrifuged for 15 min at 3,000 \u00d7 g, and the pellet was suspended in an equal volume of 50 mM NaH2PO4 buffer (pH 7.0). Cell disruption was carried out by using a sonification unit (Sonoplus HD 70; Bandelin, Berlin, Germany), with monitoring of progress under the microscope. The cell debris was removed by centrifugation for 20 min at 23,700 relative centrifugal force, and the remaining cell lysate was stored at 4\u00b0C. During fermentation of Bacillus strain SF, samples of 50 ml were taken every 30 min and were treated similarly. Assay for azoreductase activity. Assays were carried out in cuvettes (path length = 1 cm) with a total volume of 1 ml. All substances were dissolved in 50 mM phosphate buffer (pH 7.0) at 50\u00b0C and degassed with N2 for 5 min. Four hundred microliters of phosphate buffer was mixed with 200 \u03bcl of sample and 200 \u03bcl of Reactive Black 5 (100 mg liter\u20131), resulting in a final concentration of 16.3 \u03bcM dye. The reaction was started by the addition of 200 \u03bcl of NADH (7.09 mg ml\u20131; final concentration, 2 mM) and was monitored photometrically at 597 nm. The slope of the initial linear decrease of absorption (\u0394A min\u20131) was used to calculate the azoreductase activity based on the molar absorption coefficient of Reactive Black 5 (\u03b5 = 35.5 mmol\u20131 cm\u20131). One nanokatal was defined as the reduction of 1 nmol of dye per s. The same assay was used to determine the effects of inhibitors which were added to the phosphate buffer to give the concentrations indicated below. Dye decolorization in the presence of FAD was measured similarly by the addition of a 1.0 mM FAD solution to 50 mM phosphate buffer (pH 7.0) to give a final concentration of 0.2 mM FAD in the assay. The degradation of Mordant Black 9 and model dyes for the determination of degradation products and substrate specificities, respectively, was carried out as described above, with 20 \u03bcM dye and 1 mM NADH. pH and temperature optima and stability of the azoreductase. For determination of the pH dependence of azoreductase, the buffer used for the assay was set to pH values of 5.0 to 8.0. For pH values 8.5 and 9.0, a 50 mM Na2CO3-NaHCO3 buffer system was used. NADH solution and dyestuff solution were prepared with the corresponding buffers. For the determination of the temperature optimum, all solutions were brought to the corresponding temperature before mixing and the spectrophotometer was temperature controlled during measurement. Enzyme stability was tested at three different pH values and three different temperatures. For this test, 1.5 ml of enzyme solution was mixed with 4.5 ml of buffer and incubated in a thermo-controlled water bath. Samples were taken periodically, immediately frozen at \u201320\u00b0C, and subsequently analyzed. Buffers used for incubation were as follows: at pH 7.0, Na2HPO4-KH2PO4; at pH 8.5, NaBO3-NaOH-HCl; at pH 10.0, Na2CO3-NaHCO3. Oxygen measurement. For determination of the oxygen dependence of the azoreductase, a thermo-constant mini oxygen gauge (Rank Brothers Ltd.) was used. The volume of the reaction vessel was 1.5 ml and the volumes for the azoreductase assay were adjusted accordingly. The device was calibrated with a saturated Na2SO3 solution (0% O2 saturation) and by 10 min of aeration (100% O2 saturation). All reactions were carried out in 50 mM phosphate buffer at pH 7.0 at 50\u00b0C, with 2.0 mM NADH, 0.2 mM Reactive Black 5, and 0.2 mM FAD. Protein purification. The cell lysate of Bacillus strain SF was subjected to fractionated ammonium sulfate precipitation at 40% saturation to remove impurities, followed by 70% saturation in a second step to precipitate the azoreductase. This was carried out by the addition of 72.6 g of (NH4)2SO4 to 300 ml of cell lysate. The precipitated proteins after the second precipitation step were collected by centrifugation, and the pellet was dissolved in 300 ml of phosphate buffer (50 mM, pH 7.0). The solution was desalted by dialysis against phosphate buffer (50 mM, pH 7.0) overnight, until a conductivity of <10 mS cm\u20131 was measured (WTW microprocessor conductivity meter LF 96). Thereafter, the solution was concentrated by ultrafiltration in an Amicon stirred cell (model 8400; pressurized with N2 at 2 \u00d7 105 N/m2) equipped with a cellulose-polysulfonate membrane with a 10-kDa cutoff. For anion-exchange fractionation, 9.5-ml aliquots of the resulting solution were applied to a DEAE-cellulose DE 52 column installed in an \u00c4KTA fast-performance liquid chromatography system (Amersham Pharmacia Biotech). Proteins were eluted at 6 ml min\u20131 with the sample buffer and a linear gradient from 0 to 1 M NaCl. Azoreductase active fractions were pooled (approximately 50 ml) and desalted for affinity chromatography. Two-milliliter aliquots of the sample were fractionated by affinity chromatography through a HiTrap Blue affinity column from Amersham Pharmacia Biotech. The column contained Cibacron Blue F3G-A immobilized on 6% highly cross-linked spherical agarose (Sepharose High Performance) by the triazine method, binding specifically, e.g., enzymes requiring adenylyl-containing cofactors (including NADH and NADPH), albumin, coagulation factors, and interferon. Proteins were eluted at 1 ml min\u20131 with the sample buffer and a linear gradient from 0 to 1 M NaCl. Fractions with azoreductase activity were pooled and concentrated with Centricon 10 concentrators equipped with cellulose-polysulfonate membranes with a 10-kDa cutoff. The protein content of samples was measured by the Bradford method. Samples (160 \u03bcl) were pipetted into separate microtiter plate wells, and 40 \u03bcl of the reagent (obtained from Bio-Rad) per sample was added and mixed thoroughly. After 5 to 10 min of incubation at room temperature, the absorbances were measured. Bovine serum albumin was used for calibration. Gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was carried out as described previously (20), with some modifications for native PAGE, i.e., both the detergent component sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (\u03b2-mercaptoethanol) were omitted. Twelve percent polyacrylamide gels were cast, and broad-range molecular weight markers from Pharmacia were used as standards. For activity staining, carboxymethylcellulose (CMC) was mixed into the separating gel at a concentration of 0.2% (wt/vol) to enable the dye Reactive Black 5 to bind to the gel. For both SDS-PAGE and native PAGE, the Coomassie blue R-250 staining method was used. If visualization was not satisfactory, the silver staining procedure was subsequently applied. Staining of CMC native PAGE gels was done by the following procedure. CMC native gels were removed from the electrophoresis chamber and glass plates. The gel was immediately immersed in approximately 50 ml of 50 \u03bcM Reactive Black 5 solution for 25 min. After removal of the staining solution, the gel was put into a desiccator and a vacuum was applied. Subsequently, the desiccator was scavenged with N2, and a degassed NADH solution (2 mM NADH in 50 mM phosphate buffer, pH 7.0) was added under the N2 atmosphere. To make sure that no oxygen remained in the test vessel, vacuum generation and N2 scavenging were repeated alternately several times. Bands of the active enzyme appeared after approximately 10 min as white spots within a blue background. Isoelectric focusing was carried out as described elsewhere (14). Synthesis of model dyes. Anilines with substitutions of trifluoromethyl-, methyl-, chloro-, bromo-, and fluoro- groups at the o-, m-, and p- positions and 2,7-dihydroxynaphtaline-3,6-disulfonic acid disodium salt were supplied by Sigma Aldrich. Urea, sodium nitrite, and sodium hydroxide were supplied by Merck, and 32% hydrochloric acid was purchased from Riedel de Ha\u00ebn. Model azo dyes were synthesized from the corresponding aromatic amines in two steps as described elsewhere (2). Precipitation of the dyes was facilitated by acidification with hydrochloric acid. For purification, the dyes were recrystallized from hot water and freeze-dried. Determination of dye content. For confirmation of the purity of dyes, a method for the determination of the dye content was developed based on the reduction of azo bonds by excesses of sodium dithionite. Five milliliters of an oxygen-free solution of sodium dithionite (approximately 30 mM, prepared freshly every day) was mixed with 15 ml of a 100 mM formaldehyde solution to yield a more stable hydroxymethanesulfinate (26) after complete conversion. This solution was titrated with I2-KI titrimetric solution (50 mM), with soluble starch as an indicator, resulting in the consumption of about 6 ml of the solution. For determination of the normality of the I2-KI solution, 25 g of pure KI was dissolved in about 40 ml of deionized H2O to which was added 12.7 g of I2. The flask was closed and shaken until the iodine was completely dissolved. Only then was the volumetric flask filled up to exactly 1 liter, giving an approximate concentration of 0.5 M. The exact normality was determined by titration with a solution of As2O3 buffered with 2 g of sodium hydrogen-carbonate liter\u20131 in which 1 ml of the iodine solution was equivalent to 4.946 mg of As2O3. The blue complex of iodine with starch was used to indicate the end point of the titration. During titration, the complete conversion of Na2S2O4 was only found in the presence of an excess of the dye. However, extrapolation of the straight line of absorbance versus concentration of Na2S2O4 to an absorbance of 0.0 (complete reduction of the dyes) gave the exact amount of dyestuff in the sample. The molar absorption coefficients (\u03b5) of the dyes were determined by measuring the absorbance at the wavelength of maximum absorbance (\u03bbmax) of each dye. The slope of the referring trend line gives the molar absorption coefficient. HPLC and MS analysis. FAD measurements were done based on the method of Wahlund (34) in a Kontron 422S high-performance liquid chromatography (HPLC) apparatus equipped with a Hypersil RP 18 column with methanol-H2O (30:70 [vol/vol]) and 5 mmol of tetrabutylammonium hydrogen sulfate (TBAHS) as the mobile phase at a flow rate of 1 ml min\u20131 at 40\u00b0C. FAD was detected at 260 and 450 nm (\u03bbmax of FAD) with a Kontron UV 430 detector. Dye degradation products were separated in a Dionex HPLC system (Dionex Corporation, Sunnyvale, Calif.) consisting of a P580 pump, an ASI-100 automated sample injector, and a PDA-100 photodiode array detector. Chromatographic separation was performed through a Hypersil ODS 5-\u03bcm (250 by 4 mm [inside diameter]) column. The following operating conditions were used for all analyses: injection volume, 20 \u03bcl; column temperature, 40\u00b0C; mobile phase A, 100% H2O; mobile phase B, 50% methanol\u201350% H2O; gradient at 0 to 20 min, 100% (A) to 50% (B); gradient at 20 to 25 min, 50% (B); gradient at 25 to 26 min, 50 to 0% (B); gradient at 26 to 35 min, 100% (A); flow rate, 0.9 ml/min; detection wavelength, 530 nm. Mass spectral (MS) analysis was performed in an SL ion trap mass spectrometer (1100 Series LC/MSD Trap; Agilent, Waldbronn, Germany) equipped with an electrospray ionization source and connected to the HPLC system described above. The spectra were recorded in negative ionization mode. Full-scan spectra were collected from m/z 50 to 1,200, with a capillary temperature of 350\u00b0C. The nebulizer was set to 70.00 lb/in2, the dry gas was set to 12.00 liter min\u20131, and the HV capillary was set to 3,000 V. Extracted ion chromatograms were obtained by selecting ions (M-Na) at e.g., m/z 359 for Mordant Black 9.",
    "LLM": "MATERIALS AND METHODS Screening of dye-decolorizing microorganisms. Three Bacillus sp. isolates (Ba\u00adcillus sp. strain SF, Bacillus sp. strain LF, and Bacillus pallidus), previously isolated from a wastewater drain of a textile finishing company (27), were incu\u00adbated on petri dishes for 1 week at 50\u00b0C with the following medium (sterile): 250 ml of tap water, 9.25 g of standard I nutrient agar (Merck KGaA), 1.05 g of NaHCO3, and 10 mg of dye. The pH was controlled to 7.0 and 9.5, and growth and decolorization were monitored visually. Further experiments were carried out in 300-ml baffled Erlenmeyer flasks on a rotary shaker at 65\u00b0C and 160 rpm. The cultivation medium consisted of the following (in grams per liter): yeast extract (Merck), 3.0; peptone from casein (Merck), 3.0 NaHCO3, 4.2; KH2PO4, 1.0; and 1 ml of trace element solution SL-6 (29) per liter. The pH was adjusted to 9.0 and the dye concentration was 0.1 g l1. Flasks containing 90 ml of medium were inoculated with 10 ml of microorganism suspension from precultures, and dyes were added from stock solutions. The dyes Mordant Black 9 and Mordant Black 96 were from Dyestar (Frankfurt, Germany), Acid Blue 225 and Disperse Red 86 were from Ciba (Basel, Switzerland), and all other dyes were from Sigma. All dye names are given according to the color index (http://www.colour-index.org) when structures are available. Growth was monitored under a microscope, and dye degradation was measured by spectrophotometric determination of the absorbance at the wavelength maximum for each dye. Blanks were run by adding 100 mg of thiomersal [sodium ethyl-(2-mercaptobenzoato-(2)-O,S)-mercurate] per liter to prevent growth, taking into consideration the possible adsorption of the dyestuff on biomass. Cultivation of Bacillus sp. strain SF. Bacillus strain SF was grown in 300-ml baffled Erlenmeyer flasks on a rotary shaker at 60\u00b0C and 160 rpm in a medium consisting of the following components (in grams per liter): KH2PO4, 3.5; Na2HPO4 \u0001 7H2O, 7.5; yeast extract (Merck), 10.0; peptone from casein (Merck), 20.0; NH4SO4, 2.5; MgSO4 \u0001 7H2O, 4.5; MnSO4 \u0001 H2O, 0.2; iron citrate \u0001 5H2O, 0.7; and 2.5% (vol/vol) SL-6 trace element solution, as described previously (29). SL-6 trace element solution contained the following (in milligrams per liter): ZnSO4 \u0001 7H2O, 100.0; MnCl2 \u0001 4H2O, 30.0; H3BO3, 300.0; CuCl2 \u0001 2H2O, 10.0; NiCl2 \u0001 6.0H2O, 20.0; Na2MoO4 \u0001 2H2O, 30.0; and CoCl2 \u0001 6H2O, 200.0. To avoid precipitation of salts during sterilization in an autoclave, MnSO4 \u0001 H2O, MgSO4 \u0001 7H2O, and iron citrate \u0001 5H2O were autoclaved separately, and the two solutions were combined sterilely after they were cooled in a laminar flow hood. Ten-liter fermentations were carried out in a Chemap-CF 2000 fermentor, using the cultivation medium described above. The fermentor was equipped with pH and oxygen controls. Precultures were grown in baffled Erlenmeyer flasks as described above. Downstream processing. Cells were harvested at the end of the exponential phase of growth and centrifuged for 15 min at 3,000 \u0004 g, and the pellet was suspended in an equal volume of 50 mM NaH2PO4 buffer (pH 7.0). Cell disrup\u00adtion was carried out by using a sonification unit (Sonoplus HD 70; Bandelin, Berlin, Germany), with monitoring of progress under the microscope. The cell debris was removed by centrifugation for 20 min at 23,700 relative centrifugal force, and the remaining cell lysate was stored at 4\u00b0C. During fermentation of Bacillus strain SF, samples of 50 ml were taken every 30 min and were treated similarly. Assay for azoreductase activity. Assays were carried out in cuvettes (path length \u0005 1 cm) with a total volume of 1 ml. All substances were dissolved in 50 mM phosphate buffer (pH 7.0) at 50\u00b0C and degassed with N2 for 5 min. Four hundred microliters of phosphate buffer was mixed with 200 \u0006l of sample and 200 \u0006l of Reactive Black 5 (100 mg liter1), resulting in a final concentration of 16.3 \u0006M dye. The reaction was started by the addition of 200 \u0006l of NADH (7.09 mg ml1; final concentration, 2 mM) and was monitored photometrically at 597 nm. The slope of the initial linear decrease of absorption (\u0007A min1) was used to calculate the azoreductase activity based on the molar absorption coefficient of Reactive Black 5 (\u03b5 \u0005 35.5 mmol1 cm1). One nanokatal was defined as the reduction of 1 nmol of dye per s. The same assay was used to determine the effects of inhibitors which were added to the phosphate buffer to give the concentrations indicated below. Dye decolorization in the presence of FAD was measured similarly by the addition of a 1.0 mM FAD solution to 50 mM phosphate buffer (pH 7.0) to give a final concentration of 0.2 mM FAD in the assay. The degradation of Mordant Black 9 and model dyes for the determination of degradation products and substrate specificities, respectively, was carried out as described above, with 20 \u0006M dye and 1 mM NADH. pH and temperature optima and stability of the azoreductase. For determi\u00adnation of the pH dependence of azoreductase, the buffer used for the assay was set to pH values of 5.0 to 8.0. For pH values 8.5 and 9.0, a 50 mM Na2CO3- NaHCO3 buffer system was used. NADH solution and dyestuff solution were prepared with the corresponding buffers. For the determination of the temper\u00adature optimum, all solutions were brought to the corresponding temperature before mixing and the spectrophotometer was temperature controlled during measurement. Enzyme stability was tested at three different pH values and three different temperatures. For this test, 1.5 ml of enzyme solution was mixed with 4.5 ml of buffer and incubated in a thermo-controlled water bath. Samples were taken periodically, immediately frozen at 20\u00b0C, and subsequently analyzed. Buffers used for incubation were as follows: at pH 7.0, Na2HPO4-KH2PO4; at pH 8.5, NaBO3-NaOH-HCl; at pH 10.0, Na2CO3-NaHCO3. Oxygen measurement. For determination of the oxygen dependence of the azoreductase, a thermo-constant mini oxygen gauge (Rank Brothers Ltd.) was used. The volume of the reaction vessel was 1.5 ml and the volumes for the azoreductase assay were adjusted accordingly. The device was calibrated with a saturated Na2SO3 solution (0% O2 saturation) and by 10 min of aeration (100% O2 saturation). All reactions were carried out in 50 mM phosphate buffer at pH 7.0 at 50\u00b0C, with 2.0 mM NADH, 0.2 mM Reactive Black 5, and 0.2 mM FAD. Protein purification. The cell lysate of Bacillus strain SF was subjected to fractionated ammonium sulfate precipitation at 40% saturation to remove im\u00adpurities, followed by 70% saturation in a second step to precipitate the azore\u00adductase. This was carried out by the addition of 72.6 g of (NH4)2SO4 to 300 ml of cell lysate. The precipitated proteins after the second precipitation step were collected by centrifugation, and the pellet was dissolved in 300 ml of phosphate buffer (50 mM, pH 7.0). The solution was desalted by dialysis against phosphate buffer (50 mM, pH 7.0) overnight, until a conductivity of \b10 mS cm1 was measured (WTW microprocessor conductivity meter LF 96). Thereafter, the solution was concentrated by ultrafiltration in an Amicon stirred cell (model 8400; pressurized with N2 at 2 \u0004 105 N/m2) equipped with a cellulose-polysul\u00adfonate membrane with a 10-kDa cutoff. For anion-exchange fractionation, 9.5-ml aliquots of the resulting solution were applied to a DEAE-cellulose DE 52 column installed in an \u0007KTA fast\u00adperformance liquid chromatography system (Amersham Pharmacia Biotech). Proteins were eluted at 6 ml min1 with the sample buffer and a linear gradient from 0 to 1 M NaCl. Azoreductase active fractions were pooled (approximately 50 ml) and desalted for affinity chromatography. Two-milliliter aliquots of the sample were fractionated by affinity chromatog\u00adraphy through a HiTrap Blue affinity column from Amersham Pharmacia Bio\u00adtech. The column contained Cibacron Blue F3G-A immobilized on 6% highly cross-linked spherical agarose (Sepharose High Performance) by the triazine method, binding specifically, e.g., enzymes requiring adenylyl-containing cofac\u00adtors (including NADH and NADPH), albumin, coagulation factors, and inter\u00adferon. Proteins were eluted at 1 ml min1 with the sample buffer and a linear gradient from 0 to 1 M NaCl. Fractions with azoreductase activity were pooled and concentrated with Centricon 10 concentrators equipped with cellulose-poly\u00adsulfonate membranes with a 10-kDa cutoff. The protein content of samples was measured by the Bradford method. Sam\u00adples (160 \u0006l) were pipetted into separate microtiter plate wells, and 40 \u0006l of the reagent (obtained from Bio-Rad) per sample was added and mixed thoroughly. After 5 to 10 min of incubation at room temperature, the absorbances were measured. Bovine serum albumin was used for calibration. Gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was carried out as described previously (20), with some modifications for native PAGE, i.e., both the detergent component sodium dodecyl sulfate (SDS) and 2-mercapto\u00adethanol ( -mercaptoethanol) were omitted. Twelve percent polyacrylamide gels were cast, and broad-range molecular weight markers from Pharmacia were used as standards. For activity staining, carboxymethylcellulose (CMC) was mixed into the separating gel at a concentration of 0.2% (wt/vol) to enable the dye Reactive Black 5 to bind to the gel. For both SDS-PAGE and native PAGE, the Coo\u00admassie blue R-250 staining method was used. If visualization was not satisfactory, the silver staining procedure was subsequently applied. Staining of CMC native PAGE gels was done by the following procedure. CMC native gels were removed from the electrophoresis chamber and glass plates. The gel was immediately immersed in approximately 50 ml of 50 \u0006M Reactive Black 5 solution for 25 min. After removal of the staining solution, the gel was put into a desiccator and a vacuum was applied. Subsequently, the desiccator was scavenged with N2, and a degassed NADH solution (2 mM NADH in 50 mM phosphate buffer, pH 7.0) was added under the N2 atmo\u00adsphere. To make sure that no oxygen remained in the test vessel, vacuum generation and N2 scavenging were repeated alternately several times. Bands of the active enzyme appeared after approximately 10 min as white spots within a blue background. Isoelectric focusing was carried out as described elsewhere (14). Synthesis of model dyes. Anilines with substitutions of trifluoromethyl-, meth\u00adyl-, chloro-, bromo-, and fluoro- groups at the o-, m-, and p- positions and 2,7-dihydroxynaphtaline-3,6-disulfonic acid disodium salt were supplied by Sigma Aldrich. Urea, sodium nitrite, and sodium hydroxide were supplied by Merck, and 32% hydrochloric acid was purchased from Riedel de Ha\u00a8en. Model azo dyes were synthesized from the corresponding aromatic amines in two steps as described elsewhere (2). Precipitation of the dyes was facilitated by acidification with hydrochloric acid. For purification, the dyes were recrystallized from hot water and freeze-dried. Determination of dye content. For confirmation of the purity of dyes, a method for the determination of the dye content was developed based on the reduction of azo bonds by excesses of sodium dithionite. Five milliliters of an oxygen-free solution of sodium dithionite (approximately 30 mM, prepared freshly every day) was mixed with 15 ml of a 100 mM formaldehyde solution to yield a more stable hydroxymethanesulfinate (26) after complete conversion. This solution was ti\u00adtrated with I2-KI titrimetric solution (50 mM), with soluble starch as an indicator, resulting in the consumption of about 6 ml of the solution. For determination of the normality of the I2-KI solution, 25 g of pure KI was dissolved in about 40 ml of deionized H2O to which was added 12.7 g of I2. The flask was closed and shaken until the iodine was completely dissolved. Only then was the volumetric flask filled up to exactly 1 liter, giving an approximate con\u00adcentration of 0.5 M. The exact normality was determined by titration with a solution of As2O3 buffered with 2 g of sodium hydrogen-carbonate liter1 in which 1 ml of the iodine solution was equivalent to 4.946 mg of As2O3. The blue complex of iodine with starch was used to indicate the end point of the titration. During titration, the complete conversion of Na2S2O4 was only found in the presence of an excess of the dye. However, extrapolation of the straight line of absorbance versus concentration of Na2S2O4 to an absorbance of 0.0 (complete reduction of the dyes) gave the exact amount of dyestuff in the sample. The molar absorption coefficients (\u03b5) of the dyes were determined by measur\u00ading the absorbance at the wavelength of maximum absorbance ( max) of each dye. The slope of the referring trend line gives the molar absorption coefficient. HPLC and MS analysis. FAD measurements were done based on the method of Wahlund (34) in a Kontron 422S high-performance liquid chromatography (HPLC) apparatus equipped with a Hypersil RP 18 column with methanol-H2O (30:70 [vol/vol]) and 5 mmol of tetrabutylammonium hydrogen sulfate (TBAHS) as the mobile phase at a flow rate of 1 ml min1 at 40\u00b0C. FAD was detected at 260 and 450 nm ( max of FAD) with a Kontron UV 430 detector. Dye degradation products were separated in a Dionex HPLC system (Dionex Corporation, Sunnyvale, Calif.) consisting of a P580 pump, an ASI-100 auto\u00admated sample injector, and a PDA-100 photodiode array detector. Chromato\u00adgraphic separation was performed through a Hypersil ODS 5-\u0006m (250 by 4 mm [inside diameter]) column. The following operating conditions were used for all analyses: injection volume, 20 \u0006l; column temperature, 40\u00b0C; mobile phase A, 100% H2O; mobile phase B, 50% methanol\u201350% H2O; gradient at 0 to 20 min, 100% (A) to 50% (B); gradient at 20 to 25 min, 50% (B); gradient at 25 to 26 min, 50 to 0% (B); gradient at 26 to 35 min, 100% (A); flow rate, 0.9 ml/min; detection wavelength, 530 nm. Mass spectral (MS) analysis was performed in an SL ion trap mass spectrometer (1100 Series LC/MSD Trap; Agilent, Waldbronn, Germany) equipped with an electrospray ionization source and connected to the HPLC system described above. The spectra were recorded in negative ionization mode. Full-scan spectra were collected from m/z 50 to 1,200, with a capillary temperature of 350\u00b0C. The nebulizer was set to 70.00 lb/in2, the dry gas was set to 12.00 liter min1, and the HV capillary was set to 3,000 V. Extracted ion chromatograms were obtained by selecting ions (M-Na) at e.g., m/z 359 for Mordant Black 9."
  },
  "18791262": {
    "bleu": 93.85775862068965,
    "meteor": 92.38500982504519,
    "rouge1_p": 0.9471419791026429,
    "rouge1_r": 0.9494762784966112,
    "rouge1_f": 0.9483076923076923,
    "rouge2_p": 0.897908979089791,
    "rouge2_r": 0.9001233045622689,
    "rouge2_f": 0.8990147783251232,
    "rougeL_p": 0.9465273509526736,
    "rougeL_r": 0.9488601355514479,
    "rougeL_f": 0.9476923076923077,
    "cosine_similarity": 0.8743025216836569,
    "precision": 0.8563829787234043,
    "recall": 0.8563829787234043,
    "ner_f1": 0.8563829787234043,
    "ner_tp": 161,
    "ner_fp": 27,
    "ner_fn": 27,
    "bertscore_p": 0.963904082775116,
    "bertscore_r": 0.9653868675231934,
    "bertscore_f1": 0.9647872447967529,
    "bertscore_scibert_error": "The expanded size of the tensor (2407) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2407].  Tensor sizes: [1, 512]",
    "len_gt": 9474,
    "len_jl": 9473,
    "GT": "Materials and Methods E. faecalis ATCC 27274 was grown in Brain Heart Infusion (BHI) medium (Difco Laboratories, Spark, MD, USA). For long term storage, 16% glycerol stocks of the organism were prepared and stored at -80 \u00b0C. Methyl Red (MCB Chemicals, Norwood, Ohio, USA; molecular weight, 269.31, 2[4-(CH\u2083)\u2082NC\u2086H\u2084N:N]C\u2086H\u2084CO\u2082H, CAS registry 63451-28-5) was used as the azo dye under investigation. NAD(P)H (> 99% purity) was from Calbiochem (San Diego, CA, USA) and FMN was from Sigma (St. Louis, MO, USA). Chromatography resins used were Octyl Sepharose\u2122 4 Fast flow from Amersham Biosciences (Piscataway, NJ, USA) and DEAE Bio-Gel\u00ae agarose 100-200 mesh and Affi-Gel\u00ae Blue Gel 100-200 mesh from Bio-Rad (Hercules, CA, USA). The chromatography columns used for hydrophobic interaction chromatography and ion exchange chromatography were Flex-Column\u00ae from Kimble/Kontes (Vineland, NJ, USA). Purification of azoreductase Preparation of cell extracts: A single colony of E. faecalis grown on a BHI plate was used to inoculate 5 mL of BHI broth. The culture was grown at 37 \u00b0C for 12 h and 1 mL was transferred to 20 L of BHI broth. The 20 L culture was incubated at 37 \u00b0C until an OD600 = 0.9 (late log phase) was reached. The cells were harvested at 6000 \u00d7 g for 10 min at 4 \u00b0C. The bacterial pellet was resuspended in buffer A (50 mM potassium phosphate buffer, pH 7). Lysozyme and DNase I were added at final concentrations of 1 mg\u00b7mL\u207b\u00b9 and 10 \u03bcg\u00b7mL\u207b\u00b9 respectively, and the sample was incubated at 37 \u00b0C for 20 min. The sample was sonicated (probe diameter = 2 cm) on ice at 60% output using a Sonic 300 Dismembrator (Artek systems corporation, Farmingdale, NY, USA) for a total of 6 min with 20 s pulses and phenylmethanesulphonylfluoride (PMSF, 1 mM final concentration) was immediately added. The lysate was clarified by centrifugation at 100,000 \u00d7 g for 30 min at 4 \u00b0C and the pellet was discarded. Ammonium sulfate was added at 4 \u00b0C to the clarified cell extract to a final concentration of 0.5 M and EDTA was added to a final concentration of 0.5 mM. Note: All subsequent purification steps were carried out at 4 \u00b0C. Hydrophobic Interaction Chromatography (HIC): The sample was applied at a linear flow rate of 8.5 cm\u00b7h\u207b\u00b9 to an octyl sepharose 4 fast flow column (2.5 \u00d7 15.5 cm) that was equilibrated with buffer B (50 mM potassium phosphate buffer, pH 7; 0.5 mM EDTA; 0.5 M ammonium sulfate). After applying the sample, the column was washed with two volumes of 25 mM potassium phosphate buffer, pH 7 and the bound protein was eluted with 30% isopropanol. The eluted protein was concentrated to 20 mL and diafiltered with 200 mL of buffer C (10 mM Tris-Cl, pH 8, the pH of Tris-Cl was adjusted at 4 \u00b0C with HCl) in an ultrafiltration cell (Amicon Inc., Beverly, MA, USA) using a 10,000 NMWL membrane (Millipore Corporation, Bedford, MA, USA). Ion Exchange Chromatography (IEX): The sample was diluted with buffer C to obtain a final protein concentration of 7.5 mg\u00b7mL\u207b\u00b9 and applied at a linear flow rate of 7 cm\u00b7h\u207b\u00b9 to a DEAE Bio-gel agarose column (2.5 \u00d7 10.5 cm) which was previously equilibrated with buffer C. The column was washed with 300 mL of buffer C. The protein was eluted with 300 mL of buffer C containing a linear gradient of NaCl from 0\u2013200 mM. The fractions (6 mL each) that had the highest azoreductase activity were pooled together and concentrated to 11 mL. Affinity Chromatography: The pooled and concentrated sample was applied at a linear flow rate of 9 cm\u00b7h\u207b\u00b9 to Affi-gel Blue gel (2 \u00d7 1.5 cm) that was equilibrated with buffer C. The column was washed with 12 mL of buffer C containing 1 M NaCl. The bound protein was eluted with 12 mL of 30% isopropanol containing 3 M NaCl. The sample was concentrated and diafiltered with 100 mL of buffer A. The enzyme fraction was aliquoted and stored at \u201320 \u00b0C in 50% glycerol. Protein concentration was determined using the Bradford method with bovine serum albumin (BSA) as a standard. Azoreductase activity assay Qualitative assay: A preparative mini 5\u201312.5% gradient resolving polyacrylamide gel was prepared according to the method by Laemmli (Laemmli, 1970), without the addition of sodium dodecyl sulfate (SDS) using a Mini-PROTEAN\u00ae II apparatus (Bio-Rad Hercules, CA, USA). The protein sample from the affinity chromatography fraction (0.3 mg\u00b7mL\u207b\u00b9) was loaded in the single preparative reference well. Electrophoresis was performed at 4 \u00b0C in Tris-glycine buffer, pH 8.3 at 6 milliamps constant current. After electrophoresis, the gel was split down the middle and each half was placed in two shallow trays. To one tray, 5 mL buffer DH (Tris-Cl, pH 7.5; 0.5 mM Methyl Red; 10 \u03bcM FMN; 1 mM NADH) was added and to the other tray, 5 mL buffer DPH (Tris-Cl, pH 7.5; 0.5 mM Methyl Red; 10 \u03bcM FMN; 2 mM NADPH) was added. Each tray was incubated for 10 min at room temperature with gentle shaking to assure that solutions were evenly distributed across the gels. After the zones of clearing were observed, the center and the periphery of the clear zone on both gels were marked by a notch and the mobility of the clearing zone calculated. The gels were subsequently directly stained by Coomassie Blue R-250 and using the notch as the reference point, the mobility of the visible protein band was determined. Mobility was calculated as the ratio of the distance of center of clearing zone (protein) to the distance traveled by the tracking dye. Quantitative assay during purification: The assay was carried out in 1 mL cuvettes and contained 50 mM phosphate buffer, (pH 7.2); 20 \u03bcM Methyl Red; 20 \u03bcM FMN; 0.5 mM NADPH and an appropriate amount of enzyme (1\u2013800 \u03bcg). The reaction was initiated by adding NADPH. The enzyme activity was determined by an absorbance reduction in the first 2 min at 430 nm for Methyl Red using a UV-Visible spectrophotometer (UV-1601PC, Shimadzu, Columbia, MD, USA). The extinction coefficient for Methyl Red used was 23,360 M\u207b\u00b9 cm\u207b\u00b9. A unit (U) of azoreductase activity was defined as the amount of enzyme required to reduce 1 \u03bcmol per min of Methyl Red. Protein Identification Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF): The protein band corresponding to the zone of clearing seen in Fig. 1B was excised and washed with 50% acetonitrile/25 mM ammonium bicarbonate (pH 8.0). The sample was dehydrated with 100% acetonitrile and subsequently digested with a solution of 15 \u00b5g\u00b7mL\u207b\u00b9 sequencing-grade trypsin in 25 mM bicarbonate buffer. The peptides were extracted with 0.5% trifluoroacetic acid (TFA) mixed with \u03b1-cyano-4-hydroxycinnamic acid and analyzed in reflectron mode using a Voyager-DE\u2122 PRO Biospectrometry Workstation (PerSeptive Biosystems, Inc., USA). Spectra were deisotoped using a detection threshold that was manually adjusted to exclude spectral noise, and the result peak list was used to search the MSDB database (3,239,079 sequences) using the Mascot search engine (Matrix Science, London, UK). Search parameters included a statement of 100 ppm mass accuracy and the amino acid modifications: Met oxidation, pyroglutamate cyclization of glutamine, and acrylamide adducts of Cysteine. Capillary HPLC-electrospray ionization tandem mass spectra (HPLC-ESI-MS/MS): The HPLC-ESI-MS/MS data for the trypsin digested sample were acquired on a Thermo Fisher LTQ linear ion trap mass spectrometer fitted with a New Objective PicoView 550 nanospray interface. On-line HPLC separation of the digests was accomplished with an Eksigent NanoLC micro HPLC: column, PicoFrit\u2122 (New Objective, Woburn, MA, USA; 75 \u03bcm i.d.) packed to 10 cm with C18 adsorbent (Vydac, Grace, Deerfield, IL, USA; 218MS 5 \u03bcm, 300 \u00c5); mobile phase A, 0.5% acetic acid (HAc)/0.005% trifluoroacetic acid (TFA); mobile phase B, 90% acetonitrile/0.5% HAc/0.005% TFA; gradient 2 to 42% B in 30 min; flow rate, 0.4 \u03bcL\u00b7min\u207b\u00b9. MS conditions were: ESI voltage, 2.9 kV; isolation window for MS/MS, 3; relative collision energy, 35%; scan strategy, survey scan followed by acquisition of data dependent collision-induced dissociation (CID) spectra of the seven most intense ions in the survey scan above a set threshold. The uninterpreted CID spectra were searched against the NCBInr database [20080310 (6,298,708 sequences; 2,152,697,077 residues)] by means of Mascot (Matrix Science). Variable modifications considered for the searches included: oxidation of His, Met, and Trp; Trp conversion to kynurenin. Determination of protein identity probabilities were accomplished by Scaffold\u2122 (Proteome Software, Portland, Oregon, USA). Scaffold verifies peptide identifications assigned by SEQUEST and Mascot using the X!Tandem database searching program (Craig and Beavis, 2003). Scaffold then probabilistically validates these peptide identifications using PeptideProphet (Keller et al., 2002) and derives corresponding protein probabilities using ProteinProphet (Nesvizhskii et al., 2003). Pseudo first order kinetics To determine the apparent pseudo kinetic parameters, the concentration of one substrate was varied and the other substrates remained constant. The constant concentration for Methyl Red was 20 \u03bcM and the varying concentrations were 2\u201320 \u03bcM. For NADH, the constant was 1 mM and varying concentrations were 10\u201380 \u03bcM. For FMN, constant concentration was 20 \u03bcM and varying concentrations were 0.5\u20135 \u03bcM. For NADPH the varying concentrations were 1.5\u201310 mM. The parameters were determined from the double reciprocal plot using the KaleidaGraph software (Synergy software).",
    "LLM": "{\"methods\":\"Materials and Methods\\nE. faecalis ATCC 27274 was grown in Brain Heart Infusion (BHI) medium (Difco Laboratories, Spark, MD, USA). For long term storage, 16% glycerol stocks of the organism were prepared and stored at -80 \u00b0C. Methyl Red (MCB Chemicals, Norwood, Ohio, USA; molecular weight, 269.31, 2[4-(CH3)2NC6H4N:N]C6H4CO2H, CAS registry 63451-28-5) was used as the azo dye under investigation. NAD(P)\\nH (> 99% purity) was from Calbiochem (San Diego, CA, USA) and FMN was from Sigma (St. Louis, MO, USA). Chromatography resins used were Octyl Sepharose\u2122 4 Fast fow from Amersham Biosciences (Piscataway, NJ, USA) and DEAE Bio-Gel\u00ae agarose 100-200 mesh and Aff-Gel\u00ae Blue Gel 100-200 mesh from Bio-Rad (Hercules, CA, USA). The chromatography columns used for hydrophobic interaction chromatography and ion exchange chromatography were Flex-Column\u00ae from Kimble/Kontes (Vineland, NJ, USA) \\n\\nPurifcation of azoreductase \\nPreparation of cell extracts: A single colony of E. faecalis grown on a BHI plate was used to inoculate 5 mL of BHI broth. The culture was grown at 37 \u00b0C for 12 h and 1 mL was transferred to 20 L of BHI broth. The 20 L culture was incubated at 37\u00b0C until an OD600 = 0.9 (late log phase) was reached. The cells were harvested at 6000 x g for 10 min at 4 \u00b0C. The bacterial pellet was resuspended in buffer A (50 mM potassium phosphate buffer, pH 7).\\nLysozyme and DNase I were added at fnal concentrations of 1 mg.mL-1 and 10 \u03bcg.mL-1 respectively, and the sample was incubated at 37 \u00b0C for 20 min. The sample was sonicated (probe diameter = 2 cm) on ice at 60% output using a Sonic 300 Dismembrator (Artek systems corporation, farmindale, NY, USA) for a total of 6 min with 20 s pulses and phenylmethanesulphonylfuoride (PMSF, 1 mM fnal concentration) was immediately added. The lysate was clarifed by centrifugation at 100,000 x g for 30 min at 4 \u00b0C and the pellet was discarded. Ammonium sulfate was added at 4 \u00b0C to the clarifed cell extract to a fnal concentration of 0.5 M and EDTA was added to a fnal concentration of 0.5 mM. Note: All subsequent purifcations steps were carried out at 4\u00b0C. \\nHydrophobic Interaction Chromatography (HIC): The sample was applied at a linear fow rate of 8.5 cm.h-1 to an octyl sepharose 4 fast fow column (2.5 x 15.5 cm) that was equilibrated with buffer B (50 mM potassium phosphate buffer, pH 7; 0.5 mM EDTA; 0.5 M ammonium sulfate). After applying the sample, the column was washed with two volumes of 25 mM potassium phosphate buffer, pH 7 and the bound protein was eluted with 30% isopropanol. The eluted protein was concentrated to 20 mL and diafltrated with 200 mL of buffer C (10 mM Tris-Cl, pH 8, the pH of Tris-Cl was adjusted at 4 \u00b0C with HCl) in an ultrafltration cell (Amicon Inc., Beverly, MA, USA) using a 10,000 NMWL membrane (Millipore Corporation, Bedford, MA, USA).\\nIon Exchange Chromatography (IEX): The sample was diluted with buffer C to obtain a fnal protein concentration of 7.5 mg.mL-1 and applied at a linear fow rate of 7 cm.h-1 to a DEAE Bio-gel agarose column (2.5 x 10.5 cm) which was previously equilibrated with buffer C. The column was washed with 300 mL of buffer C. The protein was eluted with 300 mL of buffer C containing a linear gradient of NaCl from 0-200 mM. The fractions (6 mL each) that had the highest azoreductase activity were pooled together and concentrated to 11 mL. \\nAffnity Chromatography: The pooled and concentrated sample was applied at a linear fow rate of 9 cm.h-1 to Aff-gel Blue gel (2 x 1.5 cm) that was equilibrated with buffer C. The column was washed with 12 mL of buffer C containing 1 M NaCl. The bound protein was eluted with 12 mL of 30% isopropanol containing 3 M NaCl. The sample was concentrated and diafltrated with 100 mL of buffer A. The enzyme fraction was aliquoted and stored at \u2013 20 \u00b0C in 50% glycerol.\\nProtein concentration was determined using the Bradford method with bovine serum albumin (BSA) as a standard. \\nAzoreductase activity assay \\nQualitative assay: A preparative mini 5-12.5% gradient resolving polyacrylamide gel was prepared according to the method by Laemmli (Laemmli, 1970), without the addition of sodium dodecyl sulfate (SDS) using a Mini-PROTEAN\u00ae II apparatus (Bio-Rad Hercules, CA, USA). The protein sample from the affnity chromatography fraction (0.3 mg.mL-1) was loaded in the single preparative reference well. Electrophoresis was performed at 4 \u00b0C in Tris-glycine buffer, pH 8.3 at 6 milliamps constant current. After electrophoresis, the gel was split down the middle and each half was placed in two shallow trays. To one tray, 5 mL buffer DH (Tris-Cl, pH 7.5; 0.5 mM Methyl Red; 10 \u03bcM FMN; 1 mM NADH) was added and to the other tray, 5mL buffer DPH (Tris-Cl, pH 7.5; 0.5 mM Methyl Red; 10 \u03bcM FMN; 2 mM NADPH) was added. Each tray was incubated for 10 min at room temperature with gentle shaking to assure that solutions were evenly distributed across the gels. After the zones of clearing were observed, the center and the periphery of the clear zone on both gels were marked by a notch and the mobility of the clearing zone calculated. The gels were subsequently directly stained by Coomassie Blue R-250 and using the notch as the reference point, the mobility of the visible protein band was determined. Mobility was calculated as the ratio of the distance of center of clearing zone (A) or protein band (B) Distance of tracking dye\\nQuantitative assay during purifcation: The assay was carried out in 1 mL cuvettes and contained 50 mM phosphate buffer, (pH 7.2); 20 \u03bcM Methyl Red; 20 \u03bcM FMN; 0.5 mM NADPH and an appropriate amount of enzyme (1-800 \u03bcg). The reaction was initiated by adding NADPH. The enzyme activity was determined by an absorbance reduction in the frst 2 min at 430 nm for Methyl Red using a UV-Visible spectrophotometer (UV-1601PC, Shimadzu, Columbia, MD, USA). The extinction coeffcient for Methyl Red used was 23,360 M-1 cm-1. A unit (U) of azoreductase activity was defned as the amount of enzyme required to reduce 1 \u03bcmol per min of Methyl Red. \\nProtein Identifcation\\nMatrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF): The protein band corresponding to the zone of clearing seen in Fig. 1B was excised and washed with 50% acetonitrile/25mM ammonium bicarbonate (pH 8.0). The sample was dehydrated with 100% acetonitrile and subsequently digested with a solution of \\n15 \u00b5g.ml-1 sequencing-grade trypsin in 25 mM bicarbonate buffer. The peptides were extracted with 0.5% trifuoroacetic acid (TFA) mixed with \u03b1-cyano-4-hydroxycinnamic acid and analyzed in refectron mode using a Voyager-DE\u2122 PRO Biospectrometry Workstation (PerSeptive Biosystems, Inc., USA). Spectra were deisotoped using a detection threshold that was manually adjusted to exclude spectral noise, and the result peak list was used to search the MSDB database (3239079 sequences) using the Mascot search engine (Matrix Science, London, UK). Search parameters included a statement of 100 ppm mass accuracy and the amino acid modifcations: Met oxidation, pyroglutamate cyclization of glutamine, and acrylamide adducts of Cysteine. \\nCapillary HPLC-electrospray ionization tandem mass spectra (HPLC-ESI-MS/MS): The HPLC-ESI-MS/MS data for the trypsin digested sample were acquired on a Thermo Fisher LTQ linear ion trap mass spectrometer ftted with a New Objective PicoView 550 nanospray interface. On-line HPLC separation of the digests was accomplished with an Eksigent NanoLC micro HPLC: column, PicoFrit\u2122 (New Objective, Woburn, MA, USA; 75 \u03bcm i.d.) packed to 10 cm with C18 adsorbent (Vydac, Grace, Deerfeld, IL, USA; 218MS 5 \u03bcm, 300 \u00c5); mobile phase A, 0.5% acetic acid (HAc)/0.005% trifuoroacetic acid (TFA); mobile phase B, 90% acetonitrile/0.5% HAc/0.005% TFA; gradient 2 to 42% B in 30 min; fow rate, 0.4 \u03bcl.min-1. MS conditions were: ESI voltage, 2.9 kV; isolation window for MS/MS, 3; relative collision energy, 35%; scan strategy, survey scan followed by acquisition of data dependent collision-induced dissociation (CID) spectra of the seven most intense ions in the survey scan above a set threshold. The uninterpreted CID spectra were searched against the NCBInr database [20080310 (6298708 sequences; 2152697077 residues)] by means of Mascot (Matrix Science). Variable modifcations considered for the searches included: oxidation of His, Met, and Trp; Trp conversion to kynurenin. Determination of protein identity probabilities were accomplished by Scaffold\u2122 (Proteome Software, Portland, Oregon, USA). Scaffold verifes peptide identifcations assigned by SEQUEST and Mascot using the X!Tandem database searching program (Craig and Beavis, 2003). Scaffold then probabilistically validates these peptide identifcations using PeptideProphet (Keller et al., 2002) and derives corresponding protein probabilities using ProteinProphet (Nesvizhskii et al., 2003). \\nPseudo frst order kinetics\\nTo determine the apparent pseudo kinetic parameters, the concentration of one substrate was varied and the other substrates\u2019 remained constant. The constant concentration for Methyl Red was 20 \u03bcM and the varying concentrations were 2 - 20 \u03bcM. For NADH, the constant was 1 mM and varying concentrations were 10 - 80 \u03bcM. For FMN, constant concentration was 20 \u03bcM and varying concentrations were 0.5 - 5 \u03bcM. For NADPH the varying concentrations were 1.5 - 10 mM. The parameters were determined from the double reciprocal plot using the KaleidaGraph software (Synergy software).\"}"
  },
  "31967777": {
    "bleu": 98.16124469589816,
    "meteor": 97.60588185001563,
    "rouge1_p": 0.9903651115618661,
    "rouge1_r": 0.9964285714285714,
    "rouge1_f": 0.9933875890132249,
    "rouge2_p": 0.9827498731608321,
    "rouge2_r": 0.9887697805002552,
    "rouge2_f": 0.9857506361323155,
    "rougeL_p": 0.9903651115618661,
    "rougeL_r": 0.9964285714285714,
    "rougeL_f": 0.9933875890132249,
    "cosine_similarity": 0.9779217599025554,
    "precision": 0.9407894736842105,
    "recall": 0.9533333333333334,
    "ner_f1": 0.9470198675496689,
    "ner_tp": 143,
    "ner_fp": 9,
    "ner_fn": 7,
    "bertscore_p": 0.9873433113098145,
    "bertscore_r": 0.991793692111969,
    "bertscore_f1": 0.9896077513694763,
    "bertscore_scibert_error": "The expanded size of the tensor (2734) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2734].  Tensor sizes: [1, 512]",
    "len_gt": 11715,
    "len_jl": 11766,
    "GT": "METHODS Chemicals and Reagents. All chemicals for which the origin is not specified were purchased from Sigma-Aldrich. NADPH and NADH were purchased from Oriental Yeast Co. Ltd. Strains, Plasmids, and Growth Conditions. The protein sequence of the azoreductase, AzoA (Genbank: BBJ25239), described by Suzuki et al. was used to order a synthetic gene from Integrated DNA Technologies. Gene optimization was done for expression of AzoA in Escherichia coli, and the sequence of the synthetic gene is given in the SI of this manuscript and has been submitted to GenBank (MK674977). Using Golden Gate assembly, the gene was cloned into a pBAD vector generating two constructs for expressing AzoA with an N-terminal fusion partner: (a) AzoA fused with His-tagged SUMO (His6-SUMO), resulting in SUMO\u2212AzoA, and (b) AzoA fused to His-tagged phosphite dehydrogenase (His6-PTDH), resulting in PTDH\u2212AzoA. Primers and plasmid maps are available upon request. Plasmid sequences were verified by sequencing (Eurofins Genomics). Recombinant SUMO\u2212AzoA and PTDH\u2212AzoA were overexpressed and purified according to the following procedure: E. coli NEB10\u03b2 cells were freshly transformed with the corresponding plasmid, and an overnight culture was diluted 50-fold into 400 mL of Terrific Broth medium with 50 \u03bcg/mL ampicillin (TBamp) in 2 L baffled flasks. Cells were induced at OD600 = 1.5 with 0.02% w/v L-arabinose (final concentration) and incubated at 25 \u00b0C for 40 h (135 rpm). Cells were harvested at 4 \u00b0C and centrifuged at 4000g for 20 min. They were resuspended in 50 mM TRIS/HCl buffer pH 8.0 containing 0.5 M NaCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), disrupted by sonication in an ice/water bath and centrifuged at 4 \u00b0C 19800g for 60 min. The cell-free extract was applied in a 5 mL Ni-Sepharose FF gravity column pre-equilibrated in 50 mM TRIS/HCl buffer at pH 8.0 with 0.5 M NaCl. Stepwise elution was used to wash nonspecifically bound proteins, and elution of SUMO\u2212AzoA and PTDH\u2212AzoA was achieved with 50 mM TRIS/HCl at pH 8.0, 0.5 M NaCl, and 500 mM imidazole. Purified SUMO\u2212AzoA (in further text referred to as AzoA) and PTDH\u2212AzoA were desalted into 50 mM TRIS/HCl at pH 8.0 using EconoPac desalting columns (Bio Rad) and flash frozen in liquid nitrogen. Samples were stored at \u221270 \u00b0C until further use. The concentration of purified FMN-containing AzoA was determined based on the wild-type enzyme extinction coefficient at 461 nm (11.1 mM\u22121 cm\u22121), which was determined as previously described using trichloroacetic acid as a denaturing agent. Proteolytic digestion of the SUMO-tag was done to prepare an AzoA sample for crystallization. SUMO\u2212AzoA (2 mL, 10 mg mL\u22121) was incubated with SUMO protease (1 mg mL\u22121) overnight at 4 \u00b0C on a nutating shaker and then for 3 h at RT. The reaction mixture was applied on a pre-equilibrated Ni-Sepharose column, and the flow-through fraction, containing cleaved AzoA, was collected. Nondigested SUMO\u2212AzoA, cleaved SUMO-tag, and SUMO-protease were then eluted with 50 mM TRIS/HCl at pH 8.0 containing 0.5 M NaCl and 500 mM imidazole. All fractions were analyzed using SDS-PAGE (12% gel). Thermostability Assays. The ThermoFAD method was used to determine the apparent melting temperature of SUMO\u2212AzoA at different pH values or in the presence of different additives. Using an RT-PCR machine (CFX96-Touch, Bio-Rad), the fluorescence of the flavin cofactor was monitored using a 450\u2212490 excitation filter and a 515\u2212530 nm emission filter, typically used for SYBR Green based RT-PCR. The temperature was increased 0.5 \u00b0C per step, starting at 25 \u00b0C and ending at 90 \u00b0C, using a holding time of 10 s at each step. The maximum of the first derivative of the observed flavin fluorescence was taken as the apparent melting temperature. Twenty microliters of the enzyme solution containing 10 \u03bcM SUMO\u2212AzoA and the additive (solvent or buffer of appropriate pH) was put into a 96-well PCR plate and covered with a transparent adhesive film; then the ThermoFAD assay was run. Determination of Oligomerization State. After digestion with SUMO protease and removal of protease and His6-SUMO tag, the purified AzoA samples were concentrated to 10 mg mL\u22121. A 500 \u03bcL sample was injected into a Superdex75 10/300 column (GE Healthcare) connected to an \u00c4kta purifier (GE Healthcare). A calibration curve was made using a commercial gel permeation standard (Bio Rad). Dynamic light scattering (DLS) measurements were performed in a 5 \u03bcL disposable cuvette using a DynaPro NanoStar instrument (Wyatt Technology). The DLS results were analyzed with dynamics software, version 7 (Wyatt Technology). Steady State Kinetic Analysis. Kinetic assays were performed in duplicate for 60 s using a Jasco V-660 spectrophotometer. Different concentrations of substrates (0.01\u22120.5 mM) were dissolved in 50 mM TRIS/HCl at pH 8.5. The final concentration of methanol in the reactions was kept below 1% (v/v). AzoA (final concentration 0.01\u22120.05 \u03bcM) was added to the reaction mixture, and the reaction was started by the addition of 0.15 mM NADH (final concentration). NADH consumption (\u03b5340 = 6220 M\u22121 cm\u22121) was followed at 340 nm in the case of quinones, while reduction of indigo carmine was followed at 610 nm using \u03b5610 = 19 400 M\u22121 cm\u22121 and reduction of \u03b1-naphthol orange (Orange I) was followed at 500 nm using \u03b5500 = 20 900 M\u22121 cm\u22121. All data were analyzed using the software GraphPad Prism 6.05 (La Jolla, CA, USA). Rapid Kinetics Analysis. The catalytic and kinetic mechanism of AzoA was investigated using a SX20 stopped-flow spectrophotometer in single-mixing mode (Applied Photophysics). Absorbance spectra and single wavelength traces were acquired with a photodiode array and photomultiplier detection, respectively. The software Pro-Data 4.2.12.0 (Applied Photophysics) or GraphPad Prism 6.05 was used to analyze all data. Assays were run in duplicate or triplicate by mixing equal volumes of two reactants, at 25 \u00b0C. All solutions were prepared in 50 mM TRIS/HCl at pH 8.5. For anaerobic experiments, 100% nitrogen was bubbled through the substrate solutions (2.0 mL) containing 5.0 mM glucose. Next, 5.0 \u03bcL of glucose oxidase (Aspergillus niger, type VII, Sigma-Aldrich) was added (0.30 \u03bcM, final concentration) to remove traces of residual dioxygen. The enzyme was made anaerobic by the same procedure, except that nitrogen was blown on the surface of the solution. The flow circuit of the stopped-flow instrument was deoxygenated by repeated washes with nitrogen-bubbled buffer containing 5.0 mM glucose and 0.30 \u03bcM glucose oxidase. To study AzoA reduction using NADH as an electron donor, the enzyme (10.5 \u03bcM) was mixed with various NADH concentrations (50\u22121000 \u03bcM) in the stopped-flow cell under anaerobic conditions. Absorbance traces at 461 nm were recorded and fit to an appropriate exponential function to determine the observed rates (kobs). A plot of the kobs values versus NADH concentrations was fit to a rectangular hyperbola to calculate the pseudo-first-order rate constant for anaerobic enzyme-bound FMN reduction under saturation conditions (kred) and the apparent macroscopic dissociation constant for binding of the substrate to the enzyme (Kd). Similarly, near stoichiometric amounts of enzyme and NADH were mixed (21 and 24 \u03bcM, respectively) in the stopped-flow cell to record consecutive absorbance spectra (300\u2212700 nm) during the reaction. The reoxidation of anaerobically reduced AzoA was studied using the following potential electron acceptors: dioxygen, indigo carmine, azorubine, reactive black 5, menadione, 1,2-naphthoquinone, ferricyanide, olsalazine, and tropaeolin. First, reduced enzyme was prepared in a vial under anaerobic conditions (2.0 mL), by adding a small aliquot (<10 \u03bcL) of NADH. Stoichiometric or near stoichiometric concentrations of the reactants were used, as indicated for each experiment. The resulting solution was mixed in the stopped-flow cell with an electron acceptor to observe the resulting spectral changes. When dioxygen was used as electron acceptor, the desired final dioxygen concentrations (0.13, 0.31, 0.61, and 0.96 mM) were achieved as previously described. For all experiments, the stopped-flow traces at an appropriate wavelength were fit to an exponential function to determine the kobs values. The second-order rate constant for the reaction of dioxygen was calculated from the slope of the linear plot of kobs as a function of dioxygen concentration. Crystallization and X-ray Analysis. A concentrated sample of AzoA (24 mg mL\u22121), prepared by digestion with SUMO protease and gel permeation (vide supra), was used for crystallization experiments. Different sparse matrix screens from Molecular Dimensions (JCSG+, LMB, BCS, PACT) were tested by dispensing the conditions and the protein sample using a Mosquito robot (TTP Labtech) in 96-well two-drop Swissci plates for sitting drop (Molecular Dimensions). Drops were set at 5:3 and 3:5 ratios of a protein and reservoir mix. The protein solution was supplemented with 1.0 mM FMN and 1.0 mM NAD+. Multiple positive hits were found after 1\u22122 days of storage at 20 \u00b0C, and they were reproduced by setting handmade crystallization experiments using the same protein concentration and a 1:1 ratio for protein/reservoir. Crystals were reproduced in VDX plates for hanging drop crystallization (Hampton research). The most reproducible condition was found to be the D9 of the JCSG+ screen, containing 0.17 M ammonium sulfate and 25.2% (w/v) PEG 4000. Plates were prepared ranging in concentrations of 0.1\u22120.2 M ammonium sulfate and 20\u221230% (w/v) PEG 4000. AzoA crystals, grown in the conditions described above, were harvested and shortly passed in a cryoprotectant solution identical to the reservoir mix and supplemented with 20% glycerol. The harvested crystals were directly mounted on the goniometer of the in-house diffractometer (MarDTB Goniostat system) for preliminary characterization and data collection. Images were collected with the detector distance set at 140 mm, corresponding to a maximal theoretical resolution of 1.79 \u00c5 for our in-house setup. For one crystal, 600 images were collected at a \u0394\u03a6 of 0.3 per image, for a total of 180\u00b0. The collected images were indexed and integrated in iMosflm and the so generated .mtz file used to run aimless from the CCP4 suite for scaling. The scaled .mtz file was used in phaser together with the .pdb 3W7A as a search model for structure solution (55% identity). The structure of 3W7A was previously prepared by running chainsaw to keep only the side chains of conserved residues. The model was refined by running refmac5, alternated to manual fitting in coot. Site-Directed Mutagenesis. The Agilent primer design tool (www.agilent.com ) was used to design the primers to create mutants W60A, W60T, W60D, W60F, and W60R using QuikChange site-directed mutagenesis. Oligonucleotide sequences are available upon request. Two primers were used in each PCR reaction, using the PfuUltra II Master Mix (Agilent) as recommended by the supplier. The pBad-SUMO\u2212AzoA construct was used as a template which results in expression of AzoA mutants with a N-terminal 6xHis\u2212SUMO tag. Mutants were expressed and purified as described above for the wild-type enzyme. Phylogenetic Analysis. A phylogenetic tree of AzoA and other enzymes harboring the flavodoxin-like structure was constructed using the Neighbor-joining method. A bootstrap consensus tree was prepared, which was inferred from 500 replicates. Table S2 shows the GenBank codes for the 34 amino acid sequences selected for these analyses. The alignment required for the phylogenetic tree was generated by ClustalW. All analyses were performed using the MEGA7 software.",
    "LLM": "\u25a0 METHODS Chemicals and Reagents. All chemicals for which the origin is not specified were purchased from Sigma-Aldrich. NADPH and NADH were purchased from Oriental Yeast Co. Ltd. Strains, Plasmids, and Growth Conditions. The protein sequence of the azoreductase, AzoA (Genbank: BBJ25239), described by Suzuki et al. was used to order a synthetic gene from Integrated DNA Technologies.15 Gene optimization was done for expression of AzoA in Escherichia coli, and the sequence of the synthetic gene is given in the SI of this manuscript and has been submitted to GenBank (MK674977). Using Golden Gate assembly, the gene was cloned into a pBAD vector generating two constructs for expressing AzoA with an N-terminal fusion partner: (a) AzoA fused with His-tagged SUMO (His6-SUMO), resulting in SUMO\u2212AzoA, and (b) AzoA fused to His-tagged phosphite dehydrogenase (His6-PTDH), resulting in PTDH\u2212AzoA. Primers and plasmid maps are available upon request. Plasmid sequences were verified by sequencing (Eurofins Genomics). Recombinant SUMO\u2212AzoA and PTDH\u2212AzoA were overexpressed and purified according to the following procedure: E. coli NEB10\u03b2 cells were freshly transformed with the corresponding plasmid, and an overnight culture was diluted 50-fold into 400 mL of Terrific Broth medium with 50 \u03bcg/mL ampicillin (TBamp) in 2 L baffled flasks. Cells were induced at OD600 = 1.5 with 0.02% w/v L-arabinose (final concentration) and incubated at 25 \u00b0C for 40 h (135 rpm). Cells were harvested at 4 \u00b0C and centrifuged at 4000g for 20 min. They were resuspended in 50 mM TRIS/HCl buffer pH 8.0 containing 0.5 M NaCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), disrupted by sonication in an ice/water bath and centrifuged at 4 \u00b0C 19800g for 60 min. The cell-free extract was applied in a 5 mL Ni-Sepharose FF gravity column pre-equilibrated in 50 mM TRIS/HCl buffer at pH 8.0 with 0.5 M NaCl. Stepwise elution was used to wash nonspecifically bound proteins, and elution of SUMO\u2212AzoA and PTDH\u2212AzoA was achieved with 50 mM TRIS/HCl at pH 8.0, 0.5 M NaCl, and 500 mM imidazole. Purified SUMO\u2212AzoA (in further text referred to as AzoA) and PTDH\u2212AzoA were desalted into 50 mM TRIS/HCl at pH 8.0 using EconoPac desalting columns (Bio Rad) and flash frozen in liquid nitrogen. Samples were stored at \u221270 \u00b0C until further use. The concentration of purified FMN-containing AzoA was determined based on the wild-type enzyme extinction coefficient at 461 nm (11.1 mM\u22121 cm\u22121), which was determined as previously described using trichloroacetic acid as a denaturing agent.33 Proteolytic digestion of the SUMO-tag was done to prepare an AzoA sample for crystallization. SUMO\u2212AzoA (2 mL, 10 mg mL\u22121) was incubated with SUMO protease (1 mg mL\u22121) overnight at 4 \u00b0C on a nutating shaker and then for 3 h at RT. The reaction mixture was applied on a pre-equilibrated Ni-Sepharose column, and the flow\u2212 through fraction, containing cleaved AzoA, was collected. Non\u2212 digested SUMO\u2212AzoA, cleaved SUMO-tag, and SUMO-protease were then eluted with 50 mM TRIS/HCl at pH 8.0 containing 0.5 M NaCl and 500 mM imidazole. All fractions were analyzed using SDS\u2212 PAGE (12% gel). Thermostability Assays. The ThermoFAD method16 was used to determine the apparent melting temperature of SUMO\u2212AzoA at different pH values or in the presence of different additives. Using an RT-PCR machine (CFX96-Touch, Bio-Rad), the fluorescence of the flavin cofactor was monitored using a 450\u2212490 excitation filter and a 515\u2212530 nm emission filter, typically used for SYBR Green based RT-PCR. The temperature was increased 0.5 \u00b0C per step, starting at 25 \u00b0C and ending at 90 \u00b0C, using a holding time of 10 s at each step. The maximum of the first derivative of the observed flavin fluorescence was taken as the apparent melting temperature. Twenty microliters of the enzyme solution containing 10 \u03bcM SUMO\u2212AzoA and the additive (solvent or buffer of appropriate pH) was put into a 96-well PCR plate and covered with a transparent adhesive film; then the ThermoFAD assay was run. Determination of Oligomerization State. After digestion with SUMO protease and removal of protease and His6-SUMO tag, the purified AzoA samples were concentrated to 10 mg mL\u22121. A 500 \u03bcL sample was injected into a Superdex75 10/300 column (GE Healthcare) connected to an A\u0308kta purifier (GE Healthcare). A calibration curve was made using a commercial gel permeation standard (Bio Rad). Dynamic lights scattering (DLS) measurements were performed in a 5 \u03bcL disposable cuvette using a DynaPro NanoStar instrument (Wyatt Technology). The DLS results were analyzed with dynamics software, version 7 (Wyatt Technology). Steady State Kinetic Analysis. Kinetic assays were performed in duplicate for 60 s using a Jasco V-660 spectrophotometer. Different concentrations of substrates (0.01\u22120.5 mM) were dissolved in 50 mM TRIS/HCl at pH 8.5. The final concentration of methanol in the reactions was kept below 1% (v/v). AzoA (final concentration 0.01\u2212 0.05 \u03bcM) was added to the reaction mixture, and the reaction was started by the addition of 0.15 mM NADH (final concentration). NADH consumption (\u03b5340 = 6220 M\u22121 cm\u22121) was followed at 340 nm in the case of quinones, while reduction of indigo carmine was followed at 610 nm using \u03b5610 = 19 400 M\u22121 cm\u2212134 and reduction of \u03b1-naphthol orange (Orange I) was followed at 500 nm using \u03b5500 = 20 900 M\u22121 cm\u22121. All data were analyzed using the software GraphPad Prism 6.05 (La Jolla, CA, USA). Rapid Kinetics Analysis. The catalytic and kinetic mechanism of AzoA was investigated using a SX20 stopped-flow spectrophotometer in single-mixing mode (Applied Photophysics). Absorbance spectra and single wavelength traces were acquired with a photodiode array and photomultiplier detection, respectively. The software Pro-Data 4.2.12.0 (Applied Photophysics) or GraphPad Prism 6.05 was used to analyze all data. Assays were run in duplicate or triplicate by mixing equal volumes of two reactants, at 25 \u00b0C. All solutions were prepared in 50 mM TRIS/HCl at pH 8.5. For anaerobic experiments, 100% nitrogen was bubbled through the substrate solutions (2.0 mL) containing 5.0 mM glucose. Next, 5.0 \u03bcL of glucose oxidase (Aspergillus niger, type VII, Sigma-Aldrich) was added (0.30 \u03bcM, final concentration) to remove traces of residual dioxygen. The enzyme was made anaerobic by the same procedure, except that nitrogen was blown on the surface of the solution. The flow circuit of the stopped-flow instrument was deoxygenated by repeated washes with nitrogen-bubbled buffer containing 5.0 mM glucose and 0.30 \u03bcM glucose oxidase. To study AzoA reduction using NADH as an electron donor, the enzyme (10.5 \u03bcM) was mixed with various NADH concentrations (50\u22121000 \u03bcM) in the stopped-flow cell under anaerobic conditions. Absorbance traces at 461 nm were recorded and fit to an appropriate exponential function to determine the observed rates (kobs). A plot of the kobs values versus NADH concentrations was fit to a rectangular hyperbola to calculate the pseudo-first-order rate constant for anaerobic enzyme-bound FMN reduction under saturation conditions (kred) and the apparent macroscopic dissociation constant for binding of the substrate to the enzyme (Kd). Similarly, near stoichiometric amounts of enzyme and NADH were mixed (21 and 24 \u03bcM, respectively) in the stopped-flow cell to record consecutive absorbance spectra (300\u2212700 nm) during the reaction. The reoxidation of anaerobically reduced AzoA was studied using the following potential electron acceptors: dioxygen, indigo carmine, azorubine, reactive black 5, menadione, 1,2-naphthoquinone, ferricyanide, olsalazine, and tropaeolin. First, reduced enzyme was prepared in a vial under anaerobic conditions (2.0 mL), by adding a small aliquot (<10 \u03bcL) of NADH. Stoichiometric or near stoichiometric concentrations of the reactants were used, as indicated for each experiment. The resulting solution was mixed in the stopped\u2212 flow cell with an electron acceptor to observe the resulting spectral changes. When dioxygen was used as electron acceptor, the desired final dioxygen concentrations (0.13, 0.31, 0.61, and 0.96 mM) were achieved as previously described.35 For all experiments, the stopped\u2212 flow traces at an appropriate wavelength were fit to an exponential function to determine the kobs values. The second-order rate constant for the reaction of dioxygen was calculated from the slope of the linear plot of kobs as a function of dioxygen concentration. Crystallization and X-ray Analysis. A concentrated sample of AzoA (24 mg mL\u22121), prepared by digestion with SUMO protease and gel permeation (vide supra), was used for crystallization experiments. Different sparse matrix screens from Molecular Dimensions (JCSG+, LMB, BCS, PACT) were tested by dispensing the conditions and the protein sample using a Mosquito robot (TTP Labtech) in 96-well two-drop Swissci plates for sitting drop (Molecular Dimensions). Drops were set at 5:3 and 3:5 ratios of a protein and reservoir mix. The protein solution was supplemented with 1.0 mM FMN and 1.0 mM NAD+. Multiple positive hits were found after 1\u22122 days of storage at 20 \u00b0C, and they were reproduced by setting handmade crystallization experiments using the same protein concentration and a 1:1 ratio for protein/reservoir. Crystals were reproduced in VDX plates for hanging drop crystallization (Hampton research). The most reproducible condition was found to be the D9 of the JCSG+ screen, containing 0.17 M ammonium sulfate and 25.2% (w/v) PEG 4000 (Figure S6). Plates were prepared ranging in concentrations of 0.1\u2212 0.2 M ammonium sulfate and 20\u221230% (w/v) PEG 4000. AzoA crystals, grown in the conditions described above, were harvested and shortly passed in a cryoprotectant solution identical to the reservoir mix and supplemented with 20% glycerol. The harvested crystals were directly mounted on the goniometer of the in-house diffractometer (MarDTB Goniostat system) for preliminary character\u2212 ization and data collection. Images were collected with the detector distance set at 140 mm, corresponding to a maximal theoretical resolution of 1.79 \u00c5 for our in-house setup. For one crystal, 600 images were collected at a \u0394\u03a6 of 0.3 per image, for a total of 180\u00b0. The collected images were indexed and integrated in iMosflm and the so generated .mtz file used to run aimless from the CCP4 suite for scaling.36 The scaled .mtz file was used in phaser37 together with the .pdb 3W7A10 as a search model for structure solution (55% identity). The structure of 3W7A was previously prepared by running chainsaw38 to keep only the side chains of conserved residues. The model was refined by running refmac5,36 alternated to manual fitting in coot.39 Site-Directed Mutagenesis. The Agilent primer design tool (www.agilent.com) was used to design the primers to create mutants W60A, W60T, W60D, W60F, and W60R using QuikChange site\u2212 directed mutagenesis. Oligonucleotide sequences are available upon request. Two primers were used in each PCR reaction, using the PfuUltra II Master Mix (Agilent) as recommended by the supplier. The pBad-SUMO\u2212AzoA construct was used as a template which results in expression of AzoA mutants with a N-terminal 6xHis\u2212 SUMO tag. Mutants were expressed and purified as described above for the wild-type enzyme. Phylogenetic Analysis. A phylogenetic tree of AzoA and other enzymes harboring the flavodoxin-like structure was constructed using the Neighbor-joining method. A bootstrap consensus tree was prepared, which was inferred from 500 replicates. Table S2 shows the GenBank codes for the 34 amino acid sequences selected for these analyses. The alignment required for the phylogenetic tree was generated by ClustalW. All analyses were performed using the MEGA7 software.40"
  },
  "27502282": {
    "bleu": 92.31799910674408,
    "meteor": 92.71112065980533,
    "rouge1_p": 0.9537794896485315,
    "rouge1_r": 0.971078431372549,
    "rouge1_f": 0.9623512266213262,
    "rouge2_p": 0.9311175337186898,
    "rouge2_r": 0.9480137322216773,
    "rouge2_f": 0.9394896719319562,
    "rougeL_p": 0.9528165623495426,
    "rougeL_r": 0.9700980392156863,
    "rougeL_f": 0.9613796453728443,
    "cosine_similarity": 0.8841925631272973,
    "precision": 0.8857142857142857,
    "recall": 0.8333333333333334,
    "ner_f1": 0.8587257617728532,
    "ner_tp": 155,
    "ner_fp": 20,
    "ner_fn": 31,
    "bertscore_p": 0.9177064895629883,
    "bertscore_r": 0.9441298246383667,
    "bertscore_f1": 0.9311069846153259,
    "bertscore_scibert_error": "The expanded size of the tensor (2829) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2829].  Tensor sizes: [1, 512]",
    "len_gt": 12117,
    "len_jl": 12134,
    "GT": "Materials The enzymes XhoI, NdeI, DpnI, calf intestinal alkaline phosphatase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA); Pfu DNA polymerase was from Stratagene (La Jolla, CA), and oligonucleotides were from Sigma Genosys (The Woodlands, TX). E. coli strain DH5\u03b1 was purchased from Life Technologies, Inc. E. coli strain Rosetta(DE3)pLysS and the expression vector pET20b(+) were from Novagen (Madison, WI); QIAprep spin miniprep kit, QIAquick PCR purification kit, and QIAquick gel extraction kit were from Qiagen (Valencia, CA). The genomic DNA of P. aeruginosa PAO1 was a kind gift from Dr. Jim Spain, Georgia Institute of Technology, Atlanta, GA. HiTrapTM chelating HP 5-ml affinity column was from GE Healthcare, and isopropyl 1-thio-\u03b2-d-galactopyranoside was from Promega (Madison, WI). Cytochrome c from bovine heart, superoxide dismutase, nitroalkanes, and quinones were from Sigma. All other reagents used were of the highest purity commercially available. Cloning The gene pa1024 was amplified from the genomic DNA of P. aeruginosa PAO1 by PCR in the presence of 5% DMSO. The PCR protocol included an initial denaturation step at 95 \u00b0C, 20 cycles of denaturation for 45 s at 95 \u00b0C, annealing for 30 s at 56 \u00b0C (with the annealing temperature progressively decreasing by 0.2 \u00b0C at each cycle), extension for 3 min at 72 \u00b0C, and a final step for 10 min at 72 \u00b0C. The pa1024 gene amplified by PCR was purified by agarose gel extraction with the QIAquick gel extraction kit. The amplicon and the expression vector pET20b(+) were subjected to double digestion with NdeI and XhoI and purified with the QIAquick PCR purification kit. After a dephosphorylation step with 0.5 units of calf intestinal alkaline phosphatase for 30 min at 37 \u00b0C, the dephosphorylated vector was purified with the QIAquick PCR purification kit and ligated to the insert with incubation for 15 h at 16 \u00b0C with T4 DNA ligase. A volume of 5 \u03bcl of the ligation mixtures was used to transform E. coli strain DH5\u03b1. The resulting colonies grown at 37 \u00b0C on Luria-Bertani agar plates containing 50 \u03bcg/ml ampicillin were screened for the presence of the desired insert by DNA sequencing at the Cell, Protein, and DNA Core Facility at Georgia State University. The DNA sequencing confirmed the correct insertion of the gene in the plasmid vector and the absence of undesired mutations. Recombinant Expression and Purification E. coli expression strain Rosetta(DE3)pLysS transformed with the construct pET20b(+)/pa1024 was used to inoculate 90 ml of Terrific Broth containing 50 \u03bcg/ml ampicillin and 34 \u03bcg/ml chloramphenicol, which was incubated at 37 \u00b0C for 15 h. One aliquot of 8 ml of this culture was used to inoculate 1 liter of Terrific Broth containing 50 \u03bcg/ml ampicillin and 34 \u03bcg/ml chloramphenicol, which was incubated at 37 \u00b0C until it reached an optical density at 600 nm of 0.8. Isopropyl 1-thio-\u03b2-d-galactopyranoside was then added to a final concentration of 200 \u03bcm, and the culture was incubated at 18 \u00b0C for 20 h. The wet cell paste of 8.5 g, recovered by centrifugation, was resuspended in 40 ml of lysis buffer containing 10 mm imidazole, 300 mm NaCl, 10% v/v glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 mm MgCl2, 2 mg/ml lysozyme, 5 \u03bcg/ml DNase, 5 \u03bcg/ml RNase, and 20 mm sodium phosphate, pH 7.4. The resuspended cells were subjected to several cycles of sonication. The cell-free extract obtained after centrifugation at 12,000 \u00d7 g for 20 min was loaded onto a HiTrapTM chelating HP 5-ml affinity column equilibrated with 10 mm imidazole, 300 mm NaCl, 10% v/v glycerol, and 20 mm sodium phosphate, pH 7.4. After washing with 10 column volumes of equilibrating buffer, the column was treated with four intermediate steps at 50, 100, 150, and 250 mm imidazole in equilibration buffer to remove possible contaminants. PA1024 was eluted with 500 mm imidazole in equilibration buffer. The purest fractions based on SDS-PAGE analysis were pooled, dialyzed against 20 mm Tris-Cl, pH 8.0, 200 mm NaCl, 10% glycerol, and stored at \u221220 \u00b0C. Spectroscopic Studies UV-visible absorbance was recorded with an Agilent Technologies diode-array spectrophotometer model HP 8453 PC (Santa Clara, CA) equipped with a thermostated water bath in 20 mm Tris-Cl, pH 8.0, 200 mm NaCl, plus 10% glycerol at 25 \u00b0C. The extinction coefficient of purified PA1024 was determined by extracting the FMN cofactor by heat denaturation of the enzyme. After removing the denatured protein by centrifugation, the concentration of free FMN was determined spectroscopically by using \u03f5450 = 12,500 m\u22121 cm\u22121 (55). The concentration of flavin-bound active enzyme was determined by using the experimentally determined extinction coefficient \u03f5461 of 12,400 m\u22121 cm\u22121 (this study). The total protein concentration was determined using the Bradford method with bovine serum albumin as standard (56). Initial rates of enzymatic reaction were normalized for the concentration of enzyme-bound flavin. Fluorescence emission spectra were recorded in 20 mm Tris-Cl, pH 8.0, 200 mm NaCl, 10% glycerol, at 25 \u00b0C, with a Shimadzu model RF-5301 PC spectrofluorometer (Kyoto, Japan) using a 1-cm path length quartz cuvette. All fluorescence spectra were corrected with the corresponding blanks for Rayleigh and Raman scatterings. For flavin fluorescence, the sample at an FMN concentration of 3.2 \u03bcm was excited at 461 nm (447 nm for free FMN), and the emission scan was determined from 480 to 680 nm. Free FMN was obtained by boiling the native enzyme followed by centrifugation. Enzymatic Assays Reduction of the enzyme-bound flavin with NAD(P)H was carried out anaerobically with an SF-61DX2 Hi-Tech KinetAssyst high performance stopped-flow spectrophotometer (Bradford-on-Avon, UK), thermostated at 25 \u00b0C. Anaerobiosis of the instrument was obtained by overnight incubation with glucose (5 mm)/glucose oxidase (1 \u03bcm) in sodium pyrophosphate, pH 6.0. The enzyme was passed through a desalting PD-10 column equilibrated with 20 mm potassium phosphate, pH 7.0, 200 mm NaCl, transferred in a tonometer, and subjected to 20 cycles of degassing by applying vacuum and flushing with argon. The syringes containing 20 mm potassium phosphate, pH 7.0, 200 mm NaCl, as buffer, or the substrate NAD(P)H dissolved in buffer were flushed for 30 min with ultrapure argon before mounting onto the stopped-flow spectrophotometer. To ensure complete removal of traces of oxygen, glucose (2 mm) and glucose oxidase (0.5 \u03bcm) were present in the buffer, enzyme, and substrate solutions. The concentration of the substrate NAD(P)H was determined spectrophotometrically at 340 nm with the extinction coefficient 6,220 m\u22121 cm\u22121 (57). The concentration of the enzyme after mixing was 15 \u03bcm and of NAD(P)H ranged from 90 to 500 \u03bcm to maintain pseudo first-order conditions. Turnover of PA1024 with NADH was monitored with a suitable quinone in 20 mm potassium phosphate, 200 mm NaCl, pH 7.0, at 25 \u00b0C. NADH was kept at a constant saturating concentration of 100 \u03bcm. Stock solutions of quinones were prepared in 100% ethanol, except 2,6-dimethoxy-1,4-benzoquinone, which was dissolved in DMSO. The final ethanol or DMSO concentration in all reaction mixtures was kept fixed at 1% to minimize possible effects on enzymatic activity. Reaction rates were measured by following NADH consumption at 340 nm, using \u03f5340 = 6,220 m\u22121 cm\u22121 (58). In the case of 2-methyl-1,4-naphthoquinone, 343 nm was followed instead, because its oxidized and reduced forms are isosbestic at this wavelength. The coupled Cyt c reduction was monitored by measuring the increase of absorbance at 550 nm using an \u03f5550 = 29,500 m\u22121 cm\u22121 in 20 mm potassium phosphate, 200 mm NaCl, pH 7.0, at 25 \u00b0C. The final concentrations of NADH and Cyt c were saturating at 100 and 8.4 \u03bcm, respectively. For the experiments that compared the NADH oxidation versus the coupled Cyt c reduction, assays were done in triplicate in the presence and absence of 330 units of superoxide dismutase (SOD). Control reactions were run in the absence of the enzyme for all experiments. NADH oxidase activity was monitored on an Oxy-32 oxygen-monitoring system at atmospheric oxygen in 20 mm potassium phosphate, 200 mm NaCl, pH 7.0, at 25 \u00b0C, by following the initial rate of oxygen consumption. Final concentrations of NADH and the enzyme were 100 and 0.14 \u03bcm, respectively. To test whether hydrogen peroxide was produced during enzymatic turnover, the rate of oxygen consumption was measured at a fixed substrate concentration in the presence and absence of 170 units of catalase. The NMO activity assay was performed as described previously (5, 6, 13, 59), following the initial rate of oxygen consumption with a Hansatech Instruments computer-interfaced Oxy-32 oxygen-monitoring system at atmospheric oxygen, i.e. 230 \u03bcm oxygen, at 30 \u00b0C. Stock solutions of nitronates and nitroalkanes were prepared as described previously (5, 13). Enzyme concentration was 180 nm, and substrate concentration was 1 mm for P3N or 3-nitropropionic acid and 20 mm for 2-nitropropane, propyl-2-nitronate, nitroethane, or ethylnitronate. A positive control for nitronate monooxygenase activity was performed in parallel with purified Pa-NMO to a final concentration of 1.4 and 1 mm P3N as described previously (6). The azoreductase activity of PA1024 was tested, as described previously (19, 60), by monitoring the reduction of methyl red at 430 nm, using \u03f5430 = 23,360 m\u22121 cm\u22121, in 20 mm potassium phosphate, 200 mm NaCl, pH 7.0, at 25 \u00b0C. The final concentrations of NADH, methyl red, and enzyme were 100, 25, and 0.20 \u03bcm. The control reaction run in the absence of the enzyme was negligible, consistent with the non-enzymatic reaction between NADH and methyl red occurring only at low pH (61). Product Inhibition of PA1024 with NAD+ Product inhibition of PA1024 was carried out using NAD+ as the inhibitor with NADH and 5-hydroxy-1,4-naphthoquinone as substrates in 20 mm potassium phosphate, 200 mm NaCl, pH 7.0, at 25 \u00b0C. The final concentration of NADH was held fixed at 100 \u03bcm, whereas 5-hydroxy-1,4-naphthoquinone concentrations ranged from 2 to 17 \u03bcm. The concentration of NAD+ was kept fixed at 0.4, 1.2, and 3 mm, and a set in the absence of NAD+ was included. Data Analysis The steady-state kinetic parameters for the enzymatic assays were obtained from the fitting of the experimental points to the Michaelis-Menten equation for one substrate using KaleidaGraph software (Synergy Software, Reading, PA). Double-reciprocal plots were constructed for product inhibition patterns using KaleidaGraph, and global analysis was carried out using EnzFitter software (Biosoft, Cambridge, UK). Stopped-flow traces were fit with the software KinetAssyst 3 (TgK-Scientific, Bradford-on-Avon, UK) to Equation 1, which represents a double-exponential process. A represents the absorbance at 461 nm at time t; B1 and B2 are the amplitudes of the decrease in absorbance; kobs1 and kobs2 represent the observed rate constants for the change in absorbance; and C is an offset value accounting for the nonzero absorbance of the enzyme-bound reduced flavin at infinite time. graphic file with name zbc04016-5285-m01.jpg Concentration dependence of the observed rate constants for flavin reduction was analyzed with Equation 2, where S represents the concentration of organic substrate; kred is the rate constant for flavin reduction at saturating substrate concentration, and Kd is the apparent dissociation constant for substrate binding. graphic file with name zbc04016-5285-m02.jpg Bioinformatic Analysis The analysis of the protein sequence of PA1024 was performed with BLASTp (62), selecting the non-redundant protein sequence database. Multiple sequence alignments were also created with Clustal Omega (63) and Jalview 2.8 (64). To find hypothetical proteins sharing the six motifs of PA1024, the PHI-BLAST (65) feature was utilized. The modifications to the conserved motifs were designed manually based on the multiple sequence alignment generated by BLASTp, analysis of potential critical residues, and multiple PHI-BLAST reiterations of varying patterns.",
    "LLM": "Experimental Procedures Materials\u2014The enzymes XhoI, NdeI, DpnI, calf intestinal alkaline phosphatase, and T4 DNA ligase were from New Eng\ufffeland Biolabs (Ipswich, MA); Pfu DNA polymerase was from Stratagene (La Jolla, CA), and oligonucleotides were from Sigma Genosys (The Woodlands, TX). E. coli strain DH5\u0004 was purchased from Life Technologies, Inc. E. coli strain Rosetta(DE3)pLysS and the expression vector pET20b(\u0004) were from Novagen (Madison, WI); QIAprep spin miniprep kit, QIAquick PCR purification kit, and QIAquick gel extraction kit were from Qiagen (Valencia, CA). The genomic DNA of P. aeruginosa PAO1 was a kind gift from Dr. Jim Spain, Georgia Institute of Technology, Atlanta, GA. HiTrapTM chelating HP 5-ml affinity column was from GE Healthcare, and isopropyl 1-thio--D-galactopyranoside was from Promega (Madison, WI). Cytochrome c from bovine heart, superoxide dismutase, nitroalkanes, and quinones were from Sigma. All other reagents used were of the highest purity commercially available. Cloning\u2014The gene pa1024 was amplified from the genomic DNA of P. aeruginosa PAO1 by PCR in the presence of 5% DMSO. The PCR protocol included an initial denaturation step at 95 \u00b0C, 20 cycles of denaturation for 45 s at 95 \u00b0C, annealing for 30 s at 56 \u00b0C (with the annealing temperature progressively decreasing by 0.2 \u00b0C at each cycle), extension for 3 min at 72 \u00b0C, and a final step for 10 min at 72 \u00b0C. The pa1024 gene amplified by PCR was purified by agarose gel extraction with the QIAquick gel extraction kit. The ampli\ufffecon and the expression vector pET20b(\u0004) were subjected to double digestion with NdeI and XhoI and purified with the QIAquick PCR purification kit. After a dephosphorylation step with 0.5 units of calf intestinal alkaline phosphatase for 30 min at 37 \u00b0C, the dephosphorylated vector was purified with the QIAquick PCR purification kit and ligated to the insert with incubation for 15 h at 16 \u00b0C with T4 DNA ligase. A volume of 5 l of the ligation mixtures was used to transform E. coli strain DH5\u0004. The resulting colonies grown at 37 \u00b0C on Luria-Bertani agar plates containing 50 g/ml ampicillin were screened for the presence of the desired insert by DNA sequencing at the Cell, Protein, and DNA Core Facility at Georgia State Univer\ufffesity. The DNA sequencing confirmed the correct insertion of the gene in the plasmid vector and the absence of undesired mutations. Recombinant Expression and Purification\u2014E. coli expression strain Rosetta(DE3)pLysS transformed with the construct pET20b(\u0004)/pa1024 was used to inoculate 90 ml of Terrific Broth containing 50 g/ml ampicillin and 34 g/ml chloram\ufffephenicol, which was incubated at 37 \u00b0C for 15 h. One aliquot of 8 ml of this culture was used to inoculate 1 liter of Terrific Broth containing 50g/ml ampicillin and 34g/ml chloramphenicol, which was incubated at 37 \u00b0C until it reached an optical density at 600 nm of 0.8. Isopropyl 1-thio--D-galactopyranoside was then added to a final concentration of 200 M, and the culture was incubated at 18 \u00b0C for 20 h. The wet cell paste of 8.5 g, recovered by centrifugation, was resuspended in 40 ml of lysis buffer containing 10 mM imidazole, 300 mM NaCl, 10% v/v glyc\ufffeerol, 1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 2 mg/ml lysozyme, 5 g/ml DNase, 5 g/ml RNase, and 20 mM sodium phosphate, pH 7.4. The resuspended cells were sub\ufffejected to several cycles of sonication. The cell-free extract obtained after centrifugation at 12,000 \u0007 g for 20 min was loaded onto a HiTrapTM chelating HP 5-ml affinity column equilibrated with 10 mM imidazole, 300 mM NaCl, 10% v/v glyc\ufffeerol, and 20 mM sodium phosphate, pH 7.4. After washing with 10 column volumes of equilibrating buffer, the column was treated with four intermediate steps at 50, 100, 150, and 250 mM imidazole in equilibration buffer to remove possible contami\ufffenants. PA1024 was eluted with 500 mM imidazole in equilibra\ufffetion buffer. The purest fractions based on SDS-PAGE analysis were pooled, dialyzed against 20 mM Tris-Cl, pH 8.0, 200 mM NaCl, 10% glycerol, and stored at \u000520 \u00b0C. Spectroscopic Studies\u2014UV-visible absorbance was recorded with an Agilent Technologies diode-array spectrophotometer model HP 8453 PC (Santa Clara, CA) equipped with a thermo\ufffestated water bath in 20 mM Tris-Cl, pH 8.0, 200 mM NaCl, plus 10% glycerol at 25 \u00b0C. The extinction coefficient of purified PA1024 was determined by extracting the FMN cofactor by heat denaturation of the enzyme. After removing the denatured protein by centrifugation, the concentration of free FMN was determined spectroscopically by using \u0005450 \b 12,500 M\u00051 cm\u00051 (55). The concentration of flavin-bound active enzyme was determined by using the experimentally determined extinction coefficient \u0005461 of 12,400 M\u00051 cm\u00051 (this study). The total pro\ufffetein concentration was determined using the Bradford method with bovine serum albumin as standard (56). Initial rates of enzymatic reaction were normalized for the concentration of enzyme-bound flavin. Fluorescence emission spectra were recorded in 20 mM Tris\ufffeCl, pH 8.0, 200 mM NaCl, 10% glycerol, at 25 \u00b0C, with a Shi\ufffemadzu model RF-5301 PC spectrofluorometer (Kyoto, Japan) using a 1-cm path length quartz cuvette. All fluorescence spec\ufffetra were corrected with the corresponding blanks for Rayleigh and Raman scatterings. For flavin fluorescence, the sample at an FMN concentration of 3.2M was excited at 461 nm (447 nm for free FMN), and the emission scan was determined from 480 to 680 nm. Free FMN was obtained by boiling the native enzyme followed by centrifugation. Enzymatic Assays\u2014Reduction of the enzyme-bound flavin with NAD(P)H was carried out anaerobically with an SF-61DX2 Hi-Tech KinetAssyst high performance stopped\ufffeflow spectrophotometer (Bradford-on-Avon, UK), thermo\ufffestated at 25 \u00b0C. Anaerobiosis of the instrument was obtained by overnight incubation with glucose (5 mM)/glucose oxidase (1 M) in sodium pyrophosphate, pH 6.0. The enzyme was passed through a desalting PD-10 column equilibrated with 20 mM potassium phosphate, pH 7.0, 200 mM NaCl, transferred in a tonometer, and subjected to 20 cycles of degassing by applying vacuum and flushing with argon. The syringes containing 20 mM potassium phosphate, pH 7.0, 200 mM NaCl, as buffer, or the substrate NAD(P)H dissolved in buffer were flushed for 30 min with ultrapure argon before mounting onto the stopped\ufffeflow spectrophotometer. To ensure complete removal of traces of oxygen, glucose (2 mM) and glucose oxidase (0.5 M) were present in the buffer, enzyme, and substrate solutions. The con\ufffecentration of the substrate NAD(P)H was determined spectro\ufffephotometrically at 340 nm with the extinction coefficient 6,220 M\u00051 cm\u00051 (57). The concentration of the enzyme after mixing was 15 M and of NAD(P)H ranged from 90 to 500 M to main\ufffetain pseudo first-order conditions. Turnover of PA1024 with NADH was monitored with a suit\ufffeable quinone in 20 mM potassium phosphate, 200 mM NaCl, pH 7.0, at 25 \u00b0C. NADH was kept at a constant saturating concen\ufffetration of 100 M. Stock solutions of quinones were prepared in 100% ethanol, except 2,6-dimethoxy-1,4-benzoquinone, which was dissolved in DMSO. The final ethanol or DMSO concen\ufffetration in all reaction mixtures was kept fixed at 1% to minimize possible effects on enzymatic activity. Reaction rates were mea\ufffesured by following NADH consumption at 340 nm, using \u0005340 \b 6,220 M\u00051 cm\u00051 (58). In the case of 2-methyl-1,4-naphthoqui\ufffenone, 343 nm was followed instead, because its oxidized and reduced forms are isosbestic at this wavelength. The coupled Cyt c reduction was monitored by measuring the increase of absorbance at 550 nm using an \u0005550 \b 29,500 M\u00051 cm\u00051 in 20 mM potassium phosphate, 200 mM NaCl, pH 7.0, at 25 \u00b0C. The final concentrations of NADH and Cyt c were saturating at 100 and 8.4 M, respectively. For the experiments that compared the NADH oxidation versus the coupled Cyt c reduction, assays were done in triplicate in the presence and absence of 330 units of superoxide dismutase (SOD). Control reactions were run in the absence of the enzyme for all experiments. NADH oxidase activity was monitored on an Oxy-32 oxy\ufffegen-monitoring system at atmospheric oxygen in 20 mM potas\ufffesium phosphate, 200 mM NaCl, pH 7.0, at 25 \u00b0C, by following the initial rate of oxygen consumption. Final concentrations of NADH and the enzyme were 100 and 0.14 M, respectively. To test whether hydrogen peroxide was produced during enzy\ufffematic turnover, the rate of oxygen consumption was measured at a fixed substrate concentration in the presence and absence of 170 units of catalase. The NMO activity assay was performed as described pre\ufffeviously (5, 6, 13, 59), following the initial rate of oxygen con\ufffesumption with a Hansatech Instruments computer-inter\ufffefaced Oxy-32 oxygen-monitoring system at atmospheric oxygen, i.e. 230 M oxygen, at 30 \u00b0C. Stock solutions of nitro\ufffenates and nitroalkanes were prepared as described previ\ufffeously (5, 13). Enzyme concentration was 180 nM, and sub\ufffestrate concentration was 1 mM for P3N or 3-nitropropionic acid and 20 mM for 2-nitropropane, propyl-2-nitronate, nitroethane, or ethylnitronate. A positive control for nitro\ufffenate monooxygenase activity was performed in parallel with purified Pa-NMO to a final concentration of 1.4 and 1 mM P3N as described previously (6). The azoreductase activity of PA1024 was tested, as described previously (19, 60), by monitoring the reduction of methyl red at 430 nm, using \u0005430 \b 23,360 M\u00051 cm\u00051 , in 20 mM potassium phosphate, 200 mM NaCl, pH 7.0, at 25 \u00b0C. The final concentrations of NADH, methyl red, and enzyme were 100, 25, and 0.20 M. The control reaction run in the absence of the enzyme was negligible, consistent with the non-enzymatic reaction between NADH and methyl red occurring only at low pH (61). Product Inhibition of PA1024 with NAD\u0004\u2014Product inhibi\ufffetion of PA1024 was carried out using NAD\u0004 as the inhibitor with NADH and 5-hydroxy-1,4-naphthoquinone as substrates in 20 mM potassium phosphate, 200 mM NaCl, pH 7.0, at 25 \u00b0C. The final concentration of NADH was held fixed at 100 M, whereas 5-hydroxy-1,4-naphthoquinone concentrations ranged from 2 to 17 M. The concentration of NAD\u0004 was kept fixed at 0.4, 1.2, and 3 mM, and a set in the absence of NAD\u0004 was included. Data Analysis\u2014The steady-state kinetic parameters for the enzymatic assays were obtained from the fitting of the exper\ufffeimental points to the Michaelis-Menten equation for one substrate using KaleidaGraph software (Synergy Software, Reading, PA). Double-reciprocal plots were constructed for product inhibition patterns using KaleidaGraph, and global analysis was carried out using EnzFitter software (Biosoft, Cambridge, UK). Stopped-flow traces were fit with the soft\ufffeware KinetAssyst 3 (TgK-Scientific, Bradford-on-Avon, UK) to Equation 1, which represents a double-exponential pro\ufffecess. A represents the absorbance at 461 nm at time t; B1 and B2 are the amplitudes of the decrease in absorbance; kobs1 and kobs2 represent the observed rate constants for the change in absorbance; and C is an offset value accounting for the nonzero absorbance of the enzyme-bound reduced flavin at infinite time. A \u0006 B1 \u0005kobs1t \u0007 B2 \u0005kobs2t \u0007 C (Eq. 1) Concentration dependence of the observed rate constants for flavin reduction was analyzed with Equation 2, where S rep\uffferesents the concentration of organic substrate; kred is the rate constant for flavin reduction at saturating substrate concentra\ufffetion, and Kd is the apparent dissociation constant for substrate binding. kobs \u0006 kredS Kd \u0007 S (Eq. 2) Bioinformatic Analysis\u2014The analysis of the protein sequence of PA1024 was performed with BLASTp (62), selecting the non\uffferedundant protein sequence database. Multiple sequence align\ufffements were also created with Clustal Omega (63) and Jalview 2.8 (64). To find hypothetical proteins sharing the six motifs of PA1024, the PHI-BLAST (65) feature was utilized. The modifi\ufffecations to the conserved motifs were designed manually based on the multiple sequence alignment generated by BLASTp, analysis of potential critical residues, and multiple PHI-BLAST reiterations of varying patterns."
  },
  "35593146": {
    "bleu": 85.93372249366806,
    "meteor": 80.58872004133158,
    "rouge1_p": 0.8991696322657177,
    "rouge1_r": 0.9143546441495778,
    "rouge1_f": 0.9066985645933014,
    "rouge2_p": 0.8064133016627079,
    "rouge2_r": 0.8200483091787439,
    "rouge2_f": 0.8131736526946108,
    "rougeL_p": 0.8979833926453143,
    "rougeL_r": 0.9131483715319663,
    "rougeL_f": 0.9055023923444976,
    "cosine_similarity": 0.7881510616082384,
    "precision": 0.7555555555555555,
    "recall": 0.7157894736842105,
    "ner_f1": 0.7351351351351352,
    "ner_tp": 68,
    "ner_fp": 22,
    "ner_fn": 27,
    "bertscore_p": 0.8030028939247131,
    "bertscore_r": 0.8740349411964417,
    "bertscore_f1": 0.8385042548179626,
    "bertscore_scibert_error": "The expanded size of the tensor (1368) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1368].  Tensor sizes: [1, 512]",
    "len_gt": 4752,
    "len_jl": 4866,
    "GT": "Experimental Section Plasmid constructs: The construction of the plasmid pET16bP_AzoRo, pET16bP_AzoRo+FDH and pET16bP_FDH+AzoRo is described elsewhere.[9] Codon optimized and GC content adjusted gene sequences coding for the remaining AzoR enzymes were purchased as DNA strings from Thermo Fisher Scientific (Waltham, MA, USA) and cloned into a pET-24a(+) vector via Gibson cloning.[37] All vector constructs were checked for errors with sanger sequencing (Microsynth Seqlab, G\u00f6ttingen, Germany). The gene sequences, their corresponding amino acid sequences and the primer sequences can be found in Supporting information (Table S1). Cell-free protein synthesis: CFPS were performed using an in-house E. coli extract-based system and the PUREfrex2.0 system (GeneFrontier). The E. coli extract was prepared as described by Rolf et al.[38] Extract-based CFPS reactions with a reaction volume of 10 \u03bcL were performed in microtubes containing: E. coli cell-free extract amounting to 9.6 to 14.4 mgmL\u207b\u00b9 protein, 10 mM magnesium glutamate, 130 mM potassium glutamate, 1.5 mM each of 20 amino acids (except leucine), 1.25 mM leucine, 50 mM HEPES, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mgmL\u207b\u00b9 E. coli tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine, 30 mM 3-PGA, and 2% PEG-8000. CFPS with the PUREfrex2.0 system were carried out in microtubes according to the manufacturer\u2018s instructions with a reaction volume of 10 to 60 \u03bcL. In chaperone-assisted syntheses, 5% (v/v) DnaK or GroE mix (GeneFrontier) were added, respectively. All reactions were incubated in an Eppendorf\u00ae ThermoMixer\u00ae C for 4 to 20 h at 450 rpm and 20 to 37\u00b0C. Synthesized proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. For the analysis of the total protein fraction, 1.5 \u03bcL of the synthesis were diluted with 3.5 \u03bcL water and mixed with a 2\u00d7 SDS loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue). To obtain the soluble protein fraction, samples were centrifuged at 18.000\u00d7g for 10 min and treated as previously described. All samples were subsequently incubated at 95\u00b0C for 5 min. The PageRuler unstained protein ladder (Thermo Fischer Scientific, Waltham, MA, USA) was used as the marker. The gel was stained with 1\u00d7 Lumitein Protein Gel Stain (Biotium, Inc., Fremont, CA, USA) for 30 min. Afterwards, the gel was destained in water for 10 min and resulting bands were visualized using UV light. Electrophoretic transfer was performed on a Power Blotter XL (Thermo Fischer Scientific, Waltham, MA, USA) using Power Blotter Select Transfer Stacks (Thermo Fischer Scientific, Waltham, MA, USA). The gels were electroblotted for 7 min with 1.3 Amps. The membranes were rinsed three times with deionized water, blocked with blocking buffer TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) with 3% bovine serum albumin for 1 h at room temperature, and then rinsed twice with TBS-T. The membranes were incubated for 1 h with a solution of the primary antibody 6\u00d7-His Tag Monoclonal Antibody, diluted 1:2,000 in TBS-T, and then rinsed three times with TBS-T. The membranes treated with the antibody were further incubated for 1 h with a solution of the AP-conjugated secondary antibody goat anti-mouse IgG (H+L), diluted 1:10,000 in TBS-T, and then rinsed three times with TBS-T. To remove Tween-20, the membranes were rinsed with TBS (20 mM Tris, 150 mM NaCl, pH 7.6) twice. All membranes were finally stained with a 1-step NBT/BCIP substrate solution and incubated for 15 min. Screening for activity: Activity assays were performed in 96-well microplates with a total volume of 100 \u03bcL per well, consisting of 1 to 10% (v/v) CFPS-reaction solution and the assay solution with following final concentrations: 100 mM sodium phosphate buffer (pH 6), 50 \u03bcM FMN, 30 \u03bcM MR, BB, or 60 \u03bcM BQ and 150 \u03bcM NADH, NADPH, or BNAH, respectively. Reactions were incubated at 25\u00b0C in a FLUOstar\u00ae Omega multi-mode microplate reader (BMG LABTECH) and substrate degradation was followed at their respective wavelengths (MR 430 nm, BB 570 nm). Consumption of the co-substrates were also followed at their respective wavelengths (NADH 340 nm, NADPH 340 nm, and BNAH 358 nm). Calibration data can be found in Supporting information (Figures S4\u2013S6). The specific activity was defined as 1 U representing the conversion of 1 \u03bcmol MR/BB/NADH/NADPH/BNAH per min. For all experiments negative controls with CFPS mix and without a DNA template were carried out and the background activities were determined (see Supporting Information). All values stated have been adjusted for this background activities. All measurements were done in duplicates.",
    "LLM": "{\"methods\":\"Experimental Section\\nPlasmid constructs: The construction of the plasmid pET16bP_\\nAzoRo, pET16bP_AzoRo+FDH and pET16bP_FDH+AzoRo is de\ufffescribed elsewhere.[9] Codon optimized and GC content adjusted\\ngene sequences coding for the remaining AzoR enzymes were\\npurchased as DNA strings from Thermo Fisher Scientific (Waltham,\\nMA, USA) and cloned into a pET-24a(+) vector via Gibson cloning.[37]\\nAll vector constructs were checked for errors with sanger sequenc\ufffe\\ning (Microsynth Seqlab, G\u00f6ttingen, Germany). The gene sequences,\\ntheir corresponding amino acid sequences and the primer\\nsequences can be found in Supporting information (Table S1).\\n\\nCell-free protein synthesis: CFPS were performed using an in\ufffe\\nhouse E. coli extract-based system and the PUREfrex2.0 system\\n(GeneFrontier). The E. coli extract was prepared as described by Rolf\\net al.[38] Extract-based CFPS reactions with a reaction volume of\\n10 \u03bcL were performed in microtubes containing: E. coli cell-free\\nextract amounting to 9.6 to 14.4 mgmL\ufffe 1 protein, 10 mM magne\ufffe\\nsium glutamate, 130 mM potassium glutamate, 1.5 mM each of 20\\namino acids (except leucine), 1.25 mM leucine, 50 mM HEPES,\\n1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mgmL\ufffe 1 E. coli\\ntRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM\\nfolinic acid, 1 mM spermidine, 30 mM 3-PGA, and 2% PEG-8000.\\nCFPS with the PUREfrex2.0 system were carried out in microtubes\\naccording to the manufacturer\u2018s instructions with a reaction volume\\nof 10 to 60 \u03bcL. In chaperone-assisted syntheses, 5% (v/v) DnaK or\\nGroE mix (GeneFrontier) were added, respectively. All reactions\\nwere incubated in an Eppendorf\u00ae ThermoMixer\u00ae C for 4 to 20 h at\\n450 rpm and 20 to 37\u00b0C. Synthesized proteins were analyzed by\\nsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS\ufffe\\nPAGE) and western blot. For the analysis of the total protein\\nfraction, 1.5 \u03bcL of the synthesis were diluted with 3.5 \u03bcL water and\\nmixed with a 2\u00d7 SDS loading buffer (100 mM Tris- HCl pH 6.8, 4%\\nSDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol\\nblue). To obtain the soluble protein fraction, samples were\\ncentrifuged at 18.000\u00d7g for 10 min and treated as previously\\ndescribed. All samples were subsequently incubated at 95\u00b0C for\\n5 min. The PageRuler unstained protein ladder (Thermo Fischer\\nScientific, Waltham, MA, USA) was used as the marker. The gel was\\nstained with 1\u00d7 Lumitein Protein Gel Stain (Biotium, Inc., Fremont,\\nCA, USA) for 30 min. Afterwards, the gel was destained in water for\\n10 min and resulting bands were visualized using UV light. Electro\ufffe\\nphoretic transfer was performed on a Power Blotter XL (Thermo\\nFischer Scientific, Waltham, MA, USA) using Power Blotter Select\\nTransfer Stacks (Thermo Fischer Scientific, Waltham, MA, USA). The\\ngels were electroblotted for 7 min with 1.3 Amps. The membranes\\nwere rinsed three times with deionized water, blocked with\\nblocking buffer TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20,\\npH 7.6) with 3% bovine serum albumin for 1 h at room temper\ufffe\\nature, and then rinsed twice with TBS-T. The membranes were\\nincubated for 1 h with a solution of the primary antibody 6\u00d7-His\\nTag Monoclonal Antibody, diluted 1:2,000 in TBS-T, and then rinsed\\nthree times with TBS-T. The membranes treated with the antibody\\nwere further incubated for 1 h with a solution of the AP-conjugated\\nsecondary antibody goat anti-mouse IgG (H+L), diluted 1:10,000\\nin TBS-T, and then rinsed three times with TBS-T. To remove Tween\ufffe\\n20, the membranes were rinsed with TBS (20 mM Tris, 150 mM\\nNaCl, pH 7.6) twice. All membranes were finally stained with a 1-\\nstep NBT/BCIP substrate solution and incubated for 15 min.\\n\\nScreening for activity: Activity assays were performed in 96-well\\nmicroplates with a total volume of 100 \u03bcL per well, consisting of 1\\nto 10% (v/v) CFPS-reaction solution and the assay solution with\\nfollowing final concentrations: 100 mM sodium phosphate buffer\\n(pH 6), 50 \u03bcM FMN, 30 \u03bcM MR, BB, or 60 \u03bcM BQ and 150 \u03bcM NADH,\\nNADPH, or BNAH, respectively. Reactions were incubated at 25\u00b0C in\\na FLUOstar\u00ae Omega multi-mode microplate reader (BMG LABTECH)\\nand substrate degradation was followed at their respective wave\ufffe\\nlengths (MR 430 nm, BB 570 nm). Consumption of the co-substrates\\nwere also followed at their respective wavelengths (NADH 340 nm,\\nNADPH 340 nm, and BNAH 358 nm). Calibration data can be found\\nin Supporting information (Figures S4\u2013S6). The specific activity was\\ndefined as 1 U representing the conversion of 1 \u03bcmol MR/BB/\\nNADH/NADPH/BNAH per min. For all experiments negative controls\\nwith CFPS mix and without a DNA template were carried out and\\nthe background activities were determined (see Supporting\\nInformation). All values stated have been adjusted for this back\ufffe\\nground activities. All measurements were done in duplicates.\"}"
  },
  "28545445": {
    "bleu": 83.53317346123102,
    "meteor": 88.30796002549648,
    "rouge1_p": 0.792354474370113,
    "rouge1_r": 0.9989047097480832,
    "rouge1_f": 0.8837209302325582,
    "rouge2_p": 0.7904347826086957,
    "rouge2_r": 0.9967105263157895,
    "rouge2_f": 0.8816682832201745,
    "rougeL_p": 0.792354474370113,
    "rougeL_r": 0.9989047097480832,
    "rougeL_f": 0.8837209302325582,
    "cosine_similarity": 0.8544363387908765,
    "precision": 0.7904761904761904,
    "recall": 1.0,
    "ner_f1": 0.8829787234042553,
    "ner_tp": 83,
    "ner_fp": 22,
    "ner_fn": 0,
    "bertscore_p": 0.49200132489204407,
    "bertscore_r": 0.4871184527873993,
    "bertscore_f1": 0.4916074573993683,
    "bertscore_scibert_error": "The expanded size of the tensor (1888) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1888].  Tensor sizes: [1, 512]",
    "len_gt": 6021,
    "len_jl": 7738,
    "GT": "Methods Reagents Oligonucleotides were synthesised by Life Technologies (Carlsbad, CA, US). Except otherwise mentioned, all other chemicals were supplied by Sigma-Aldrich (St. Louis, MO, US). Bacterial strains and plasmids E. faecalis (EF) and Escherichia coli (EC) strains were selected from the bioM\u00e9rieux strain collection. They were isolated from human, animal or food sources and originated from different geographic areas (Table 1). E. faecalis V583 was used as matrix for the amplification of putative reductases coding genes. E. coli XL1Blue (Stratagene, San Diego, US) was host for the modified pQE30 plasmids (Qiagen, Courtaboeuf, France) used for recombinant protein overexpression (Table 2). Bacterial nitroreductase activity testing Eight E. faecalis strains and an E. coli strain as control, all part of bioM\u00e9rieux strains collection were tested for their nitroreductase activity. For each strain, 100 \u03bcL of a 1 McFarland suspension was inoculated into 100 \u03bcL of Trypcase Soy broth (bioM\u00e9rieux, France) containing 150 \u03bcM of 7-nitrocoumarin-3-carboxylic acid (7NCCA) [17] and incubated at 35 \u00b0C with shaking for 24 h. The bacterial reduction of this nitro substrate generates a fluorescent product (\u03bbex = 365 nm, \u03bbem = 440 nm). Kinetic of nitroreduction was followed on an Infinite\u00ae M200 microplate reader (TECAN, M\u00e4nnedorf, Switzerland). In silico search of nitroreductases in the E. faecalis V583 genome sequence The protein BLAST search was carried out on E. faecalis V583 published transcribed genome using two reference sequences: NfsA (NCBI reference sequence AAC73938.1.) and NfsB (AAC73679.1.), which are the two major nitroreductases in E. coli MG1655. As E. coli azoreductase AzoR displays nitroreductase activity [6, 18], a similar BLAST protein search was also performed using AzoR as the reference protein (AAC74494.1.). Phylogenetic data analyses Sequence alignments and tree constructions were done using Geneious 7.1 (http://www.geneious.com , [19]). Protein sequences were compared using Muscle alignment. Trees were constructed using neighbour-joining method and out-grouped with the NQO1 sequence, a human quinone NADH dehydrogenase (AAB60701.1). The selected sequences all represented experimentally verified bacterial azoreductases and/or nitroreductases. Cloning of targeted genes E. faecalis strain V583 DNA was used for amplification of putative nitroreductases coding genes. The plasmid pQE30 (Qiagen, Courtaboeuf, France) was used for cloning. To obtain chromosomal DNA, E. faecalis cells were lysed in a solution containing Tris (0.1 M), EDTA (0.01 M) pH 8 and lysozyme (20 mg.ml\u22121) during 30 min at 37 \u00b0C followed by addition of proteinase K (1.4 mg.ml\u22121), RNase (1.4 mg.ml\u22121) and sarcosyl solution (2%). Incubation with slow shaking was continued for another hour at 37 \u00b0C. DNA was then extracted using a phenol/chloroform/isoamylalcohol mix (V/V/V; 25/24/1) (Roth, Karlsruhe, Germany) and chloroform/isoamylacolhol (V/V; 24/1) before precipitation by cold ethanol (at 70% final concentration). The oligonucleotides used for gene amplification and cloning are listed in Table 3. PCR was carried out as described by Mercier et al. [18]. PCR products were analysed (5 \u03bcL aliquots) by agarose gel electrophoresis (1% agar in Tris-acetate-EDTA buffer) and further purified using the QIAquick purification kit (Qiagen, Courtaboeuf, France). The purified fragments and the expression vector pQE30 were digested by restriction enzymes BamHI and SalI prior to ligation. The ligation was carried out using T4 DNA ligase (Fermentas, Saint-R\u00e9my-l\u00e8s-Chevreuse, France) under standard conditions. All the constructed plasmids were verified by sequencing (GATC Biotech, Konstanz, Germany) to confirm the insertion and the absence of mutations in the sequences cloned. E. coli strain XL1Blue was used as a host strain to facilitate overproduction of the different proteins. The recombinant vectors were transformed into XL1Blue cells by electroporation. The recombinant transformants were selected by their ampicillin resistance (100 mg.l\u22121). Purification of enzymes His-tagged recombinant enzymes were purified according to two different processes previously described by Mercier et al. [18]. The native method allowed to recover enzymes including bound cofactors. A denaturation/renaturation protocol allowed the isolation of enzymes without cofactors. Excess (unbound) cofactors and imidazole used in the elution step of purification process were eliminated by dialysis. Whole cells extracts and overexpressed (and purified) recombinant proteins were analyzed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [20]. Enzymatic assays Enzymatic activities were assayed with 10 mg. l\u22121 of purified proteins and 100 \u03bcM of substrate. Methyl red and 7NCCA are used as substrate for azo and nitro activities. Reaction is followed in 50 mM sodium phosphate pH 7 buffer added with 0.5 mM NAD(P) H, in a 96-well microplate (Greiner, Courtaboeuf, France). The kinetic analyses were performed using purified proteins incubated at 35 \u00b0C while continuously measuring fluorescence development using an Infinite\u00ae M200 microplate reader. Absorbance at both excitation and emission wavelengths were quantified in order to evaluate potential quenching effects. Nitroreductase activity was evaluated by fluorescence increase at 365/440 nm (excitation/emission), corresponding to emergence of the fluorescent products of 7NCCA nitroreduction. Azoreductase activity was evaluated using methyl red as substrate. Reduction of this compound was detected by absorbance at 435 nm and by fluorescence at 250/395 nm (excitation/emission), parameters used to detect anthranilic acid. All experiments were independently reproduced three to five times. All the fluorescence results were expressed in relative units. To simplify the graph, one experiment in each case has been chosen to draw the curves but all our experiments have shown very good reproducibility",
    "LLM": "Methods Reagents Oligonucleotides were synthesised by Life Technologies (Carlsbad, CA, US). Except otherwise mentioned, all other chemicals were supplied by Sigma-Aldrich (St. Louis, MO, US). Bacterial strains and plasmids E. faecalis (EF) and Escherichia coli (EC) strains were selected from the bioM\u00e9rieux strain collection. They were isolated from human, animal or food sources and originated from different geographic areas (Table 1). E. faecalis V583 was used as matrix for the amplification of putative reductases coding genes. E. coli XL1Blue (Stratagene, San Diego, US) was host for the modified pQE30 plasmids (Qiagen, Courtaboeuf, France) used for recombinant protein overexpression (Table 2). Table 1 Strains used in the study Species Collections Code Number Escherichia coli bioM\u00e9rieux EC 76.10.041 Enterococcus faecalis bioM\u00e9rieux EF1 80.04.017 Enterococcus faecalis bioM\u00e9rieux \u2013 ATCC29212 EF2 83.11.066 Enterococcus faecalis bioM\u00e9rieux EF3 95.01.009 Enterococcus faecalis bioM\u00e9rieux EF4 98.06.158 Enterococcus faecalis bioM\u00e9rieux EF5 00.08.222 Enterococcus faecalis bioM\u00e9rieux EF6 04.05.001 Enterococcus faecalis bioM\u00e9rieux EF7 07.06.031 Enterococcus faecalis bioM\u00e9rieux \u2013 ATCC700802 V583 95.07.074 Escherichia coli bioM\u00e9rieux \u2013 Stratagene XL1Blue 13.02.209 Bacterial nitroreductase activity testing Eight E. faecalis strains and an E. coli strain as control, all part of bioM\u00e9rieux strains collection were tested for their nitroreductase activity. For each strain, 100 \u03bcL of a 1 McFarland suspension was inoculated into 100 \u03bcL of Trypcase Soy broth (bioM\u00e9rieux, France) containing 150 \u03bcM of 7-nitrocoumarin-3-carboxylic acid (7NCCA) [17] and incubated at 35 \u00b0C with shaking for 24 h. The bacterial reduction of this nitro substrate generates a fluorescent product (\u03bbex = 365 nm, \u03bbem = 440 nm). Kinetic of nitroreduction was followed on an Infinite\u00ae M200 microplate reader (TECAN, M\u00e4nnedorf, Switzerland). In silico search of nitroreductases in the E. faecalis V583 genome sequence The protein BLAST search was carried out on E. faecalis V583 published transcribed genome using two reference sequences: NfsA (NCBI reference sequence AAC73938.1.) and NfsB (AAC73679.1.), which are the two major nitroreductases in E. coli MG1655. As E. coli azoreductase AzoR displays nitroreductase activity [6, 18], a similar BLAST protein search was also performed using AzoR as the reference protein (AAC74494.1.). Phylogenetic data analyses Sequence alignments and tree constructions were done using Geneious 7.1 (http://www.geneious.com, [19]). Protein sequences were compared using Muscle alignment. Trees were constructed using neighbour-joining method and out-grouped with the NQO1 sequence, a human quinone NADH dehydrogenase (AAB60701.1). The selected sequences all represented experimentally verified bacterial azoreductases and/or nitroreductases. Cloning of targeted genes E. faecalis strain V583 DNA was used for amplification of putative nitroreductases coding genes. The plasmid pQE30 (Qiagen, Courtaboeuf, France) was used for cloning. To obtain chromosomal DNA, E. faecalis cells were lysed in a solution containing Tris (0.1 M), EDTA (0.01 M) pH 8 and lysozyme (20 mg.ml\u22121) during 30 min at 37 \u00b0C followed by addition of proteinase K (1.4 mg.ml\u22121), RNase (1.4 mg.ml\u22121) and sarcosyl solution (2%). Incubation with slow shaking was continued for another hour at 37 \u00b0C. DNA was then extracted using a phenol/chloroform/isoamylalcohol mix (V/V/V; 25/24/1) (Roth, Karlsruhe, Germany) and chloroform/isoamylacohol (V/V; 24/1) before precipitation by cold ethanol (at 70% final concentration). The oligonucleotides used for gene amplification and cloning are listed in Table 3. PCR was carried out as described by Mercier et al. [18]. PCR products were analysed (5 \u03bcL aliquots) by agarose gel electrophoresis (1% agar in Tris-acetate-EDTA buffer) and further purified using the QIAquick purification kit (Qiagen, Courtaboeuf, France). The purified fragments and the expression vector pQE30 were digested by restriction enzymes BamHI and SalI prior to ligation. The ligation was carried out using T4 DNA ligase (Fermentas, Saint-R\u00e9my-l\u00e8s-Chevreuse, France) under standard conditions. All the constructed plasmids were verified by sequencing (GATC Biotech, Konstanz, Germany) to confirm the insertion and the absence of mutations in the sequences cloned. E. coli strain XL1Blue was used as a host strain to facilitate overproduction of the different proteins. The recombinant vectors were transformed into XL1Blue cells by electroporation. The recombinant transformants were selected by their ampicillin resistance (100 mg. l\u22121). Purification of enzymes His-tagged recombinant enzymes were purified according to two different processes previously described by Mercier et al. [18]. The native method allowed to recover enzymes including bound cofactors. A denaturation/renaturation protocol allowed the isolation of enzymes without cofactors. Excess (unbound) cofactors and imidazole used in the elution step of purification process were eliminated by dialysis. Whole cells extracts and overexpressed (and purified) recombinant proteins were analyzed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [20]. Enzymatic assays Enzymatic activities were assayed with 10 mg. l\u22121 of purified proteins and 100 \u03bcM of substrate. Methyl red and 7NCCA are used as substrate for azo and nitro activities. Reaction is followed in 50 mM sodium phosphate pH 7 buffer added with 0.5 mM NAD(P) H, in a 96-well microplate (Greiner, Courtaboeuf, France). The kinetic analyses were performed using purified proteins incubated at 35 \u00b0C while continuously measuring fluorescence development using an Infinite\u00ae M200 microplate reader. Absorbance at both excitation and emission wavelengths were quantified in order to evaluate potential quenching effects. Nitroreductase activity was evaluated by fluorescence increase at 365/440 nm (excitation/emission), corresponding to emergence of the fluorescent products of 7NCCA nitroreduction. Azoreductase activity was evaluated using methyl red as substrate. Reduction of this compound was detected by absorbance at 435 nm and by fluorescence at 250/395 nm (excitation/emission), parameters used to detect anthranilic acid. All experiments were independently reproduced three to five times. All the fluorescence results were expressed in relative units. To simplify the graph, one experiment in each case has been chosen to draw the curves but all our experiments have shown very good reproducibility. Table 2 Plasmids constructed for the study Name Cloned gene DNA extracted from pQE30-azoA95 azoA Enterococcus faecalis V583 pQE30-EF0404 ef0404 Enterococcus faecalis V583 pQE30-EF0648 ef0648 Enterococcus faecalis V583 pQE30-EF0655 ef0655 Enterococcus faecalis V583 pQE30-EF1181 ef1181 Enterococcus faecalis V583 Table 3 Primers used for amplification and cloning of ef0404, ef0648, ef0655 and ef1181 in pQE30 plasmid Targeted gene Primers Tm (\u00b0C) PCR amplicon azoA cgggatccTCAAAATTATTAGTTGTTAAAGCACATCC 59.5 644 pb acgcgtcgacATTTAGAATGTTTTACCGTATTCAGTTGC 60.4 ef0404 cgggatccACAACATATACAACGAATGATTTTTCAG 59.0 660 pb acgcgtcgacTTTTATTGCCTATTCAAATGTCGTG 59.5 ef0648 cgggatccATGTATCAAGATGTTGTTCGCAGC 60.6 701 pb acgcgtcgacCAATCACTTTGGATGTTTGTTCC 58.6 ef0655 cgggatccTCAAAATTTACTGAAATGATGAAAAACC 60.1 652 pb acgcgtcgacGCTTTCACTCCTTTCCTCTTCG 59.7 ef1181 cgggatccAATCAAACAATCGAACAATTACTAAGTC 57.7 763 pb acgcgtcgacCACGCTCTTTTGTTTAGACATC 58.2 For each gene, primer couples are reported. Nucleotides identical to the gene sequence are in capital letters and nucleotide motifs required for cloning containing restriction sites BamHI or SalI are in lowercase"
  },
  "12147495": {
    "bleu": 84.24107142857142,
    "meteor": 85.89765249045661,
    "rouge1_p": 0.8406454866364095,
    "rouge1_r": 0.9893175074183976,
    "rouge1_f": 0.9089422028353327,
    "rouge2_p": 0.8289606458123108,
    "rouge2_r": 0.9756532066508313,
    "rouge2_f": 0.8963447899618112,
    "rougeL_p": 0.8396369137670197,
    "rougeL_r": 0.9881305637982196,
    "rougeL_f": 0.9078516902944385,
    "cosine_similarity": 0.8409868932855507,
    "precision": 0.6768558951965066,
    "recall": 0.9226190476190477,
    "ner_f1": 0.7808564231738035,
    "ner_tp": 155,
    "ner_fp": 74,
    "ner_fn": 13,
    "bertscore_p": 0.869642436504364,
    "bertscore_r": 0.8979196548461914,
    "bertscore_f1": 0.8841442465782166,
    "bertscore_scibert_error": "The expanded size of the tensor (2863) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2863].  Tensor sizes: [1, 512]",
    "len_gt": 10226,
    "len_jl": 11678,
    "GT": "MATERIALS AND METHODS Bacterial strains, media, and plasmids. X. azovorans KF46 was originally isolated from a soil inoculum after a prolonged enrichment with carboxy-Orange II [1-(4-carboxyphenylazo)-2-naphtol] as sole source of carbon and energy (27). For the present study, strain KF46F DSM 13620 was used, which is a nonmucoid variant of strain KF46, which had been preserved freeze-dried during the last 25 years (T. Leisinger, personal communication). X. azovorans KF46F was routinely cultivated in a mineral medium with 4-hydroxybenzoate and Orange II, supplemented with proline and a trace element solution as described previously by Zimmermann et al. (49). Escherichia coli DH5\u03b1 and E. coli BL21(DE3)pLysS were used as host strains for recombinant DNA work. E. coli strains were routinely cultured at 37\u00b0C in Luria-Bertani medium which was supplemented with ampicillin (100 \u00b5g/ml), if appropriate. The plasmid pBluescript II KS(+) (1) was used for most cloning experiments, and the plasmid vector pET11a (44) was used for high levels of expression. Preparation of cell extracts. The cells were suspended in 100 mM potassium phosphate buffer (pH 7.1) and disrupted by using a French press (Aminco, Silver Spring, Md.) at 80 or 125 MPa. Cell debris were removed by centrifugation at 100,000 \u00d7 g for 30 min at 4\u00b0C. Protein was determined by the method of Bradford (7) using bovine serum albumin as a standard. Standard assay for the determination of enzyme activities with cell extracts and purified enzyme preparations. The standard enzyme assays contained in 1 ml 87 \u00b5mol of potassium phosphate buffer (pH 7.1), 1 \u00b5mol of NADH, 8 nmol of Orange II and different amounts of protein (1 to 600 \u00b5g). The reaction was spectrophotometrically assayed at room temperature at 482 nm (\u03b5\u2084\u2088\u2082 = 18.2 mM\u207b\u00b9 cm\u207b\u00b9). One unit of enzyme activity was defined as the amount of enzyme that catalyzed the decolorization of 1 \u00b5mol of substrate per min. Conversion of different azo dyes by the azoreductase. The reaction mixtures for the determination of the substrate specificity of the azoreductase contained in 1 ml 87 \u00b5mol of potassium phosphate buffer (pH 7.1), 1 \u00b5mol of NADH, and 25 nmol of the respective azo compounds and cell extracts (0.15 mg/ml) from E. coli BL21(DE3)pLysSpET-OII-Ex9, which expressed the azoreductase from X. azovorans KF46F. The relevant wavelengths and extinction coefficients for these dyes are summarized in Table 1. Enzyme purification. Protein was purified at room temperature by use of a fast-performance liquid chromatography system consisting of an LCC 500 controller, pump P-500, UV-1 monitor, conductivity monitor, REC-482 recorder, and FRAC autosampler from Amersham Pharmacia Biotech (Uppsala, Sweden). X. azovorans KF46F was grown in a 10-liter fermentation vessel on a medium with 4-hydroxybenzoate (15 mM) and Orange II (0.2 mM) at 30\u00b0C as described above. The culture medium was intensively stirred (450 rpm). The optical density at 546 nm and the concentration of the azo dye (\u03bbmax = 482 nm) were determined spectrophotometrically. The cells were harvested by centrifugation during the exponential growth phase when the optical density reached an optical density at 546 nm of about 2 and a significant decolorization of Orange II was noticed. A crude extract was prepared using a French press, and 402 mg of protein was applied in two portions to a Red Sepharose CL-6B column (column volume, 32 ml; Amersham Pharmacia Biotech). Proteins that were not bound to the column were eluted with 0.1 M K-phosphate buffer (pH 8.5). The proteins bound to the column were subsequently eluted with 90 ml of a linear gradient of 0.1 M K-phosphate buffer into 0.1 M K-phosphate buffer plus 60 mM NADH at a flow rate of 1 to 2 ml/min. Fractions (4 ml each) were collected, and azo reductase activity was determined spectrophotometrically. The azoreductase was eluted as a single peak at a concentration of about 50 to 55 mM NADH. The active fractions were pooled (11 mg of protein, 12.2 U of azo reductase activity), and 0.5 M (NH\u2084)\u2082SO\u2084 was added. The solution was incubated for 15 min on ice and finally filtered (Minisart NML [0.2-\u00b5m pore size]; Sartorius, G\u00f6ttingen, Germany). This filtrate was transferred to an octyl-Sepharose column (column volume, 9 ml; Amersham Pharmacia Biotech). Protein was eluted with 120 ml of a linear gradient of K-phosphate buffer (0.1 M, pH 8.5) plus 0.5 M (NH\u2084)\u2082SO\u2084 into K-phosphate buffer (0.1 M, pH 8.5) plus 50% (vol/vol) ethylene glycol at a flow rate of 0.1 to 0.3 ml/min. The active fractions (5 ml each) eluted at about 45 to 50% (vol/vol) ethylene glycol (0.72 mg of protein, 3.3 U of azoreductase activity). The fractions containing azoreductase activity were concentrated by ultrafiltration (Centricon 30; Amicon, Danvers, Mass.). The concentrated sample (about 1 ml) was applied to a Superdex 75 prep grade column (Amersham Pharmacia Biotech) and eluted with 90 ml of K-phosphate buffer (0.1 M, pH 7.1) at a flow rate of 1.5 ml/min. Fractions (0.5 ml each) with azoreductase activity were pooled. Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (28). Gels were silver stained by the method of Merril et al. (31) using the Amersham Pharmacia Biotech silver stain kit. Determination of molecular weight. The relative molecular mass of the native enzyme was determined by gel filtration using a Superdex 75 prep-grade column (Amersham Pharmacia Biotech) and appropriate standard proteins. Protein cleavage, isolation of peptides, and sequencing of peptides and N termini. The digestion of the azoreductase by trypsin (Sigma, Deisenhofen, Germany) and the subsequent separation of tryptic digests by reversed-phase high-pressure liquid chromatography (HPLC) were performed as described previously (43). The digestion of the enzyme with endoproteinase Glu-C (Sigma) was performed in Na-phosphate buffer (pH 7.8) in order to ensure a proteolytic cleavage of the enzyme on the carboxyl-side of glutamate or aspartate residues. For the digestions, 38 or 23 \u00b5g, respectively, of the purified azoreductase was incubated with 1.5 \u00b5g or 1 \u00b5g of trypsin or endoproteinase C, respectively. The digests were incubated for 24 h at 37\u00b0C, and the individual peptides were purified by reverse-phase HPLC. The amino acid sequences were determined by automated Edman degradation using an Applied Biosystems model 491 sequencer. DNA manipulation techniques. The genomic DNA was prepared as described by Ausubel et al. (3). Plasmid DNA from E. coli DH5\u03b1 was isolated with the Flexi-Prep kit (Amersham Pharmacia Biotech) or the Qiaprep Spin Miniprep kit (Qiagen, Hilden, Germany). Digestion of DNA with restriction endonucleases (Gibco BRL, New England Biolabs, Frankfurt, Germany), electrophoresis, purification, and ligation with T4 DNA ligase (Gibco BRL) were performed according to the standard procedures (39). Transformation of E. coli was done by the method of Inoue et al. (20). For cloning of PCR products a T vector was prepared as described by Marchuk et al. (30). PCR. Oligonucleotides were custom synthesized according to the known or deduced sequences of the amino-terminal amino acid sequence and various internal peptides. PCR mixtures (50 \u00b5l) for the amplification of genomic DNA contained 50 pmol of each primer, 0.1 \u00b5g of genomic template DNA, a 0.1 mM concentration of each deoxynucleoside triphosphate, 0 to 7.5% (vol/vol) dimethyl sulfoxide, 1.5 mM MgCl\u2082, 0.7 U of Taq DNA polymerase, and the corresponding reaction buffer (Gibco BRL). For the amplification reaction with the primers deduced from the amino terminus and the internal peptides, the following PCR program was used: an initial denaturation (95\u00b0C, 3 min; addition of the Taq polymerase after 2 min) was followed by 29 cycles consisting of an annealing temperature of 50\u00b0C (1.5 min), a polymerization step (72\u00b0C, 2 min), and denaturation (95\u00b0C, 40 s). The last polymerization step was extended to 10 min. The PCR products were initially cloned into the T-tailed EcoRV-site of pBluescript II KS(+) (30). Hybridization procedures. A digoxygenin DNA labeling and detection kit was used according to the instructions of the supplier (Boehringer Mannheim). The hybridization temperature was set to 68\u00b0C. DNA sequencing and nucleotide sequence analysis. The DNA sequence was determined by dideoxy chain termination with double-stranded DNA of clones and overlapping subclones in an automated DNA sequencing system (ALFexpress-Sequencer; Amersham Pharmacia Biotech) with fluorescently labeled primers or nucleotides. Sequence analysis, database searches, and comparisons were done with the PCGene software package, release 6.85, and the BLAST search at the National Center for Biotechnology Information (NCBI). The alignment of the azoreductases was obtained with the program CLUSTAL using the default parameters. Expression of the azoreductase in E. coli. For expression in E. coli, azoB was inserted into pET11a (44) under the control of the phage T7 promoter. The DNA segment encompassing azoB was amplified by PCR with simultaneous introduction of an NdeI site upstream and a BamHI site downstream of azoB. The following oligonucleotide primers were used for the amplification: 5-ATG ACA TAT GAT TCT GGT CGT CGG AGG AAC-3 and 5-GCG CGG ATC CGA CGG CAT CGA GAG CAT C. The amplified products were cleaved with NdeI and BamHI and ligated into pET11a. E. coli DH5\u03b1 was transformed with the resulting plasmids. The plasmids were subsequently isolated and introduced into E. coli BL21(DE3)pLysS by transformation. Chemicals. The azo dyes and all other chemicals were obtained from Aldrich (Steinheim, Germany), Fluka (Buchs, Switzerland), Merck (Darmstadt, Germany), Sigma, and Gerbu Biotech (Gaiberg, Germany). The azo dyes Mordant Yellow 3 and 1-(4-hydroxyphenylazo)-2-naphthol-6-sulfonate were kindly provided by Bayer AG (Leverkusen, Germany) and K. Bredereck (University of Stuttgart), respectively. The oligonucleotides were synthesized by MWG Biotech (Ebersberg, Germany). Nucleotide sequence accession number. The nucleotide sequence of the 5,782-bp SstI fragment was deposited in GenBank under accession number AF466104.",
    "LLM": "MATERIALS AND METHODS Bacterial strains, media, and plasmids. X. azovorans KF46 was originally isolated from a soil inoculum after a prolonged enrichment with carboxy-Orange II [1-(4-carboxyphenylazo)-2-naphtol] as sole source of carbon and energy (27). For the present study, strain KF46F DSM 13620 was used, which is a nonmucoid variant of strain KF46, which had been preserved freeze-dried during the last 25 years (T. Leisinger, personal communication). X. azovorans KF46F was routinely cultivated in a mineral medium with 4-hydroxybenzoate and Orange II, supple\ufffemented with proline and a trace element solution as described previously by Zimmermann et al. (49). Escherichia coli DH5 and E. coli BL21(DE3)pLysS were used as host strains for recombinant DNA work. E. coli strains were routinely cultured at 37\u00b0C in Luria-Bertani medium which was supplemented with ampicillin (100 \u0004g/ml), if appropriate. The plasmid pBluescript II KS(\u0001) (1) was used for most cloning experiments, and the plasmid vector pET11a (44) was used for high levels of expression. Preparation of cell extracts. The cells were suspended in 100 mM potassium phosphate buffer (pH 7.1) and disrupted by using a French press (Aminco, Silver Spring, Md.) at 80 or 125 MPa. Cell debris were removed by centrifugation at 100,000 \u0005 g for 30 min at 4\u00b0C. Protein was determined by the method of Bradford (7) using bovine serum albumin as a standard. Standard assay for the determination of enzyme activities with cell extracts and purified enzyme preparations. The standard enzyme assays contained in 1 ml 87 \u0004mol of potassium phosphate buffer (pH 7.1), 1 \u0004mol of NADH, 8 nmol of Orange II and different amounts of protein (1 to 600 \u0004g). The reaction was spectrophotometrically assayed at room temperature at 482 nm (\u03b5482 \u0006 18.2 mM\u00071 cm\u00071 ). One unit of enzyme activity was defined as the amount of enzyme that catalyzed the decolorization of 1 \u0004mol of substrate per min. Conversion of different azo dyes by the azoreductase. The reaction mixtures for the determination of the substrate specificity of the azoreductase contained in 1 ml 87 \u0004mol of potassium phosphate buffer (pH 7.1), 1 \u0004mol of NADH, and 25 nmol of the respective azo compounds and cell extracts (0.15 mg/ml) from E. coli BL21(DE3)pLysSpET-OII-Ex9, which expressed the azoreductase from X. azovorans KF46F. The relevant wavelengths and extinction coefficients for these dyes are summarized in Table 1. Enzyme purification. Protein was purified at room temperature by use of a fast-performance liquid chromatography system consisting of an LCC 500 con\ufffetroller, pump P-500, UV-1 monitor, conductivity monitor, REC-482 recorder, and FRAC autosampler from Amersham Pharmacia Biotech (Uppsala, Sweden). X. azovorans KF46F was grown in a 10-liter fermentation vessel on a medium with 4-hydroxybenzoate (15 mM) and Orange II (0.2 mM) at 30\u00b0C as described above. The culture medium was intensively stirred (450 rpm). The optical density at 546 nm and the concentration of the azo dye (\bmax \u0006 482 nm) were deter\ufffemined spectrophotometrically. The cells were harvested by centrifugation during the exponential growth phase when the optical density reached an optical density at 546 nm of about 2 and a significant decolorization of Orange II was noticed. A crude extract was prepared using a French press, and 402 mg of protein was applied in two portions to a Red Sepharose CL-6B column (column volume, 32 ml; Amersham Pharmacia Biotech). Proteins that were not bound to the column were eluted with 0.1 M K-phosphate buffer (pH 8.5). The proteins bound to the column were subsequently eluted with 90 ml of a linear gradient of 0.1 M K-phosphate buffer into 0.1 M K-phosphate buffer plus 60 mM NADH at a flow rate of 1 to 2 ml/min. Fractions (4 ml each) were collected, and azo reductase activity was determined spectrophotometrically. The azoreductase was eluted as a single peak at a concentration of about 50 to 55 mM NADH. The active fractions were pooled (11 mg of protein, 12.2 U of azo reductase activity), and 0.5 M (NH4)2SO4 was added. The solution was incubated for 15 min on ice and finally filtered (Minisart NML [0.2-\u0004m pore size]; Sartorius, Go\u00a8ttingen, Germa\ufffeny). This filtrate was transferred to an octyl-Sepharose column (column volume, 9 ml; Amersham Pharmacia Biotech). Protein was eluted with 120 ml of a linear gradient of K-phosphate buffer (0.1 M, pH 8.5) plus 0.5 M (NH4)2SO4 into K-phosphate buffer (0.1 M, pH 8.5) plus 50% (vol/vol) ethylene glycol at a flow rate of 0.1 to 0.3 ml/min. The active fractions (5 ml each) eluted at about 45 to 50% (vol/vol) ethylene glycol (0.72 mg of protein, 3.3 U of azoreductase activity). The fractions containing azoreductase activity were concentrated by ultrafiltra\ufffetion (Centricon 30; Amicon, Danvers, Mass.). The concentrated sample (about 1 ml) was applied to a Superdex 75 prep grade column (Amersham Pharmacia Biotech) and eluted with 90 ml of K-phosphate buffer (0.1 M, pH 7.1) at a flow rate of 1.5 ml/min. Fractions (0.5 ml each) with azoreductase activity were pooled. Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (28). Gels were silver stained by the method of Merril et al. (31) using the Amersham Pharmacia Biotech silver stain kit. Determination of molecular weight. The relative molecular mass of the native enzyme was determined by gel filtration using a Superdex 75 prep-grade column (Amersham Pharmacia Biotech) and appropriate standard proteins. Protein cleavage, isolation of peptides, and sequencing of peptides and N termini. The digestion of the azoreductase by trypsin (Sigma, Deisenhofen, Germany) and the subsequent separation of tryptic digests by reversed-phase high-pressure liquid chromatography (HPLC) were performed as described pre\ufffeviously (43). The digestion of the enzyme with endoproteinase Glu-C (Sigma) was performed in Na-phosphate buffer (pH 7.8) in order to ensure a proteolytic cleavage of the enzyme on the carboxyl-side of glutamate or aspartate residues. For the digestions, 38 or 23 \u0004g, respectively, of the purified azoreductase was incubated with 1.5 \u0004g or 1 \u0004g of trypsin or endoproteinase C, respectively. The digests were incubated for 24 h at 37\u00b0C, and the individual peptides were purified by reverse-phase HPLC. The amino acid sequences were determined by auto\ufffemated Edman degradation using an Applied Biosystems model 491 sequencer. DNA manipulation techniques. The genomic DNA was prepared as described by Ausubel et al. (3). Plasmid DNA from E. coli DH5 was isolated with the Flexi-Prep kit (Amersham Pharmacia Biotech) or the Qiaprep Spin Miniprep kit (Qiagen, Hilden, Germany). Digestion of DNA with restriction endonucleases (Gibco BRL, New England Biolabs, Frankfurt, Germany), electrophoresis, pu\uffferification, and ligation with T4 DNA ligase (Gibco BRL) were performed ac\ufffecording to the standard procedures (39). Transformation of E. coli was done by the method of Inoue et al. (20). For cloning of PCR products a T vector was prepared as described by Marchuk et al. (30). PCR. Oligonucleotides were custom synthesized according to the known or deduced sequences of the amino-terminal amino acid sequence and various internal peptides. PCR mixtures (50 \u0004l) for the amplification of genomic DNA contained 50 pmol of each primer, 0.1 \u0004g of genomic template DNA, a 0.1 mM concentration of each deoxynucleoside triphosphate, 0 to 7.5% (vol/vol) dimethyl sulfoxide, 1.5 mM MgCl2, 0.7 U of Taq DNA polymerase, and the corresponding reaction buffer (Gibco BRL). For the amplification reaction with the primers deduced from the amino terminus and the internal peptides, the following PCR program was used: an initial denaturation (95\u00b0C, 3 min; addition of the Taq polymerase after 2 min) was followed by 29 cycles consisting of an annealing temperature of 50\u00b0C (1.5 min), a polymerization step (72\u00b0C, 2 min), and denaturation (95\u00b0C, 40 s). The last polymerization step was extended to 10 min. The PCR products were initially cloned into the T-tailed EcoRV-site of pBlue\ufffescript II KS(\u0001) (30). Hybridization procedures. A digoxygenin DNA labeling and detection kit was used according to the instructions of the supplier (Boehringer Mannheim). The hybridization temperature was set to 68\u00b0C. TABLE 1. Color index registration numbers, absorption maxima, purity, and calculated molar extinction coefficients of sulfonated azo dyes used in this studya Azo dye CI number Dye content (%) Absorp\ufffetion max (nm) Extinction coefficient (mM\u00071 cm\u00071 ) Acid Orange 7 (Orange II) 15510 95 482 18.2 Mordant Violet 5 (Violet N) 15670 NSb 529 10.7 Acid Orange 8 15575 65 486 27.4 Acid Orange 12 (CroceinOrange G) 15970 70 482 24.7 Acid Red 66 (Ponceau BS) 26905 NS 502 22.9 Acid Red 88 15620 75 500 7.8 Food Yellow 3 (Sunset Yellow FD6) 15985 NS 480 19.8 Solvent Yellow 14 (Sudan I) 12055 NS 557 8.7 Solvent Orange 7 (Sudan II) 12140 90 559 4.3 Solvent Red 23 (Sudan III) 26100 NS 512 2.3 Solvent Red 24 (Sudan IV) 26105 NS 515 5.7 Acid Orange 10 (Orange G) 16230 NS 477 20.7 Acid Red 18 (Neucoccin) 16255 NS 507 19.9 Acid Red 27 (Amaranth) 16185 90 520 22.6 Acid Black 52 (Palatine Fast Black WAN) 15711 25 565 63.2 Acid Red 151 26900 40 486 6.9 1-(2-Pyridylazo)-2-naphthol NS 445 7.7 Calconcarboxylic acid NS 555 3.2 Calmagite NS 539 9.9 a The dyes were purchased from Fluka, Aldrich, or Sigma and not further purified. The absorption maxima were determined in NaK phosphate buffer (pH 7.4, 54 mM). The molar extinction coefficients were calculated using the dye purities indicated. If the dye purity was not indicated by the supplier, it was assumed that the preparations consisted of pure dye. b NS, dye purity not specified by the supplier. DNA sequencing and nucleotide sequence analysis. The DNA sequence was determined by dideoxy chain termination with double-stranded DNA of clones and overlapping subclones in an automated DNA sequencing system (ALFexpress-Sequencer; Amersham Pharmacia Biotech) with fluorescently la\ufffebeled primers or nucleotides. Sequence analysis, database searches, and comparisons were done with the PCGene software package, release 6.85, and the BLAST search at the National Center for Biotechnology Information (NCBI). The alignment of the azoreduc\ufffetases was obtained with the program CLUSTAL using the default parameters. Expression of the azoreductase in E. coli. For expression in E. coli, azoB was inserted into pET11a (44) under the control of the phage T7 promoter. The DNA segment encompassing azoB was amplified by PCR with simultaneous introduction of an NdeI site upstream and a BamHI site downstream of azoB. The following oligonucleotide primers were used for the amplification: 5-ATG ACA TAT GAT TCT GGT CGT CGG AGG AAC-3 and 5-GCG CGG ATC CGA CGG CAT CGA GAG CAT C. The amplified products were cleaved with NdeI and BamHI and ligated into pET11a. E. coli DH5 was transformed with the resulting plasmids. The plasmids were subsequently isolated and introduced into E. coli BL21(DE3)pLysS by transformation. Chemicals. The azo dyes and all other chemicals were obtained from Aldrich (Steinheim, Germany), Fluka (Buchs, Switzerland), Merck (Darmstadt, Germa\ufffeny), Sigma, and Gerbu Biotech (Gaiberg, Germany). The azo dyes Mordant Yellow 3 and 1-(4-hydroxyphenylazo)-2-naphthol-6-sulfonate were kindly pro\ufffevided by Bayer AG (Leverkusen, Germany) and K. Bredereck (University of Stuttgart), respectively. The oligonucleotides were synthesized by MWG Biotech (Ebersberg, Germany). Nucleotide sequence accession number. The nucleotide sequence of the 5,782- bp SstI fragment was deposited in GenBank under accession number AF466104."
  },
  "19666717": {
    "bleu": 88.09840425531915,
    "meteor": 90.6021625572944,
    "rouge1_p": 0.8839857651245552,
    "rouge1_r": 0.9802683504340963,
    "rouge1_f": 0.9296407185628743,
    "rouge2_p": 0.8618233618233618,
    "rouge2_r": 0.9557661927330173,
    "rouge2_f": 0.9063670411985019,
    "rougeL_p": 0.8839857651245552,
    "rougeL_r": 0.9802683504340963,
    "rougeL_f": 0.9296407185628743,
    "cosine_similarity": 0.8656614087170112,
    "precision": 0.7936507936507936,
    "recall": 0.8547008547008547,
    "ner_f1": 0.823045267489712,
    "ner_tp": 100,
    "ner_fp": 26,
    "ner_fn": 17,
    "bertscore_p": 0.7996780276298523,
    "bertscore_r": 0.8382138609886169,
    "bertscore_f1": 0.8194746971130371,
    "bertscore_scibert_error": "The expanded size of the tensor (2040) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2040].  Tensor sizes: [1, 512]",
    "len_gt": 7465,
    "len_jl": 8135,
    "GT": "MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The E. coli strains and plasmids used in this study are listed in Table 1. They were grown aerobically at 37\u00b0C in Luria-Bertani (LB) medium. Construction of azoR knockout mutant. The one-step replacement method described by Datsenko and Wanner (12) was used to construct an azoR deletion in E. coli JM109. PCR was done using pKD4 as a template and primers D1 (5\u2032-TGGACTGAATATCTACAGTCCACATCAAGACCGTGTCCGGCATATGAATATCCTCCTTAG-3\u2032) and D2 (5\u2032-AACGGGGCATCGCCCATTCAAACATCTATAAGGAAACACCGTGTAGGCTGGAGCTGCTTC-3\u2032). The underlined sequences anneal to the template plasmid, while the remaining sequences correspond to the ends of azoR. The PCR-amplified DNA fragment was electroporated into E. coli ADH containing plasmid pKD46, which expresses Red recombinase and was cured later by growth at 37\u00b0C. The mutant strain E. coli LG1 was selected on an LB plate supplemented with kanamycin. The deletion of the gene was confirmed by PCR and sequencing. Cloning of azoR from E. coli JM109. The azoR gene was PCR-amplified from the chromosomal DNA of E. coli JM109 by using primers P1 (5\u2032-GAATTCATGAGCAAGGTATTAGTTCTTAA-3\u2032) and P2 (5\u2032-CTCGAGTTATGCAGAAACAATGCTGTCG-3\u2032). The amplified DNA fragment was digested with EcoRI and XhoI and cloned into the EcoRI/XhoI double-digested pGEX-4T-1. The resulting plasmid pGEX-azoR was transformed into E. coli JM109, and the resultant E. coli LG2 was used for the expression of GSH S-transferase (GST) fusion AzoR protein. Purification and reductase activity assay of AzoR. E. coli LG2 cells were grown in LB medium to an optical density at 600 nm (OD600) of 0.5, and then the azoR gene was induced by adding 0.5 mM isopropyl-\u03b2-D-thiogalactopyranoside to the cultures and further incubating them for 3 additional hours. Cells were then harvested by centrifugation at 4,000 \u00d7 g for 15 min and washed twice with 20 mM sodium phosphate buffer, pH 7.0. The pellets were frozen at \u221280\u00b0C overnight. The cells that were thawed on ice and resuspended in phosphate buffer were disrupted by ultrasonication three times for 20 s each time and spun at 50,000 \u00d7 g for 20 min at 4\u00b0C. The supernatant fraction was saved. The GST-AzoR protein was purified by glutathione Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer\u2019s instructions. The GST tag was removed from AzoR by digestion with 10 U of thrombin per mg of fusion protein. It was mixed gently and incubated in cleavage buffer (20 mM Tris-HCl [pH 8.5], 120 mM NaCl, and 2.5 mM CaCl2) at room temperature (22 to 25\u00b0C) for 16 h. The thrombin-treated samples were purified again with glutathione Sepharose 4B according to the manufacturer\u2019s instructions. The progress of AzoR purification was monitored by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein concentration was determined by the method of Bradford (5), with bovine serum albumin as a standard. The standard reductase activity assay system comprised 20 mM sodium phosphate buffer (pH 7.0), 10 to 700 \u03bcM electron acceptor substrate (methyl red or various quinone compounds), 100 \u03bcM NADH, 20 \u03bcM FMN, and a suitable amount of enzyme. The reaction was initiated by the addition of NADH. Reductase activities were monitored based on the oxidation of NADH at 340 nm (\u03b5 = 6.22 mM\u207b\u00b9 cm\u207b\u00b9) (26). Values of Km (\u03bcM) and kcat (s\u207b\u00b9) were obtained from Lineweaver-Burk plots, and kcat/Km (s\u207b\u00b9 mM\u207b\u00b9) values were calculated. Effects of stressors on growth of E. coli strains. The overnight cultures of E. coli JM109 and E. coli LG1 were used to inoculate fresh LB medium to a starting OD600 of 0.1. They were grown at 37\u00b0C aerobically until the OD600 reached 0.6. Then each culture was split equally into several portions and treated with stressors, including 0.5 mM 2-MHQ, 1 mM diamide, 6 mM catechol, 0.3 mM menadione, and 1 and 10 mM H2O2. Intracellular GSH measurements were made using a GSH assay kit (Calbiochem, San Diego, CA). To study the effects of exogenous GSH on the growth of E. coli JM109 and E. coli LG1 in the presence of 2-MHQ, 0.25 to 2 mM GSH was added to the cultures before the addition of 0.5 mM 2-MHQ. Quantitative PCR analysis. E. coli JM109 culture was grown at 37\u00b0C aerobically, and samples were taken in different growth phases to study the transcription of azoR under normal growth conditions. Effects of different stressors on the transcription of azoR were evaluated. Various stressors were applied to E. coli cultures when the OD600 reached 0.6. Samples were taken before (0 min) and 10 and 20 min after addition of stressors. Total RNA was obtained with RNAiso reagent (TaKaRa Dalian). DNA contamination was eliminated by a DNase I treatment at 37\u00b0C for 1 h. RNA integrity was checked by agarose gel electrophoresis. The concentration and purity of extracted RNA were determined by measuring the A260 and A280. In total, 0.5 \u03bcg of purified RNA was reverse transcribed (15 min at 37\u00b0C and 5 s at 85\u00b0C) by using PrimeScript real-time (RT) enzyme mix I (TaKaRa Dalian). For the reverse transcription, 50 \u03bcM Oligo dT primer and 100 \u03bcM random hexamers were used in each reaction. Quantitative RT-PCR was performed with a TaqMan assay in a Thermal Cycler Dice RT system (TaKaRa Dalian) using the following conditions: 10 s at 95\u00b0C and 45 cycles of 5 s at 95\u00b0C and 30 s at 60\u00b0C. The primers used for amplification of azoR cDNA were azoR-F (5\u2032-GGGATCCACAAAGATGGACCAAC-3\u2032) and azoR-R (5\u2032-GGCGAAGACAAATTTCACATCGG-3\u2032), and the TaqMan probe was azoR-P (5\u2032-[FAM]-ACCTGGTGACGCCGTATCTGTCCACG-[Eclipse]-3\u2032). E. coli 16S rRNA was chosen as an endogenous control for normalization of the cDNA loading in each PCR. Primers used for amplification of 16S rRNA cDNA were 16S rRNA-F (5\u2032-CCATGAAGTTGGAATCGCTAG-3\u2032) and 16S rRNA-R (5\u2032-GCTTGACGGGCGGTGT-3\u2032), and the TaqMan probe was 16S rRNA-P (5\u2032-[FAM]-TACAAGGCCCGGGAACGTATTCACCG-[Eclipse]-3\u2032). The reaction mixtures (25 \u03bcl) contained 12.5 \u03bcl of Premix Ex Taq (2\u00d7) (TaKaRa Dalian), 10 \u03bcM each primer, 3 \u03bcM TaqMan probe, and 2-\u03bcl portions of different dilutions of the reverse transcription product. The standard curves for quantification were calculated by serial 10-fold dilutions of the standard cDNA transcript. Control reaction in which reverse transcriptase was omitted from the reaction mixture ensured that DNA products resulted from the amplification of cDNA rather than from DNA contamination. Determination of TSP. RNA was extracted from cells exposed to 2-MHQ for 10 min. Determination of the transcription start point (TSP) was performed using the 5\u2032-Full rapid amplification of cDNA ends (RACE) kit (TaKaRa Dalian) according to manufacturer\u2019s instructions. The adaptor-ligated cDNA was amplified by two sequential reactions using azoR outer primer (5\u2032-CATCGCTCGGACGCAGA-3\u2032) and azoR inner primer (5\u2032-CAACCAGTTCGCCATCCAGTA-3\u2032). After denaturation at 94\u00b0C for 3 min, the amplification (30 cycles) was 98\u00b0C for 15 s, 52\u00b0C for 30 s, and 72\u00b0C for 2 min, followed by one cycle of 72\u00b0C for 10 min. The PCR products were sequenced to recognize the relevant TSP. Sequence alignment and structure analysis. Multiple sequence alignment was conducted using Clustal X 1.8 (10) and visualized with Genedoc 2.6 (32). Three-dimensional structure analysis was conducted using PyMol 0.99 (DeLano Scientific, San Carlos, CA). Statistical analysis. Student\u2019s t test was employed to investigate statistical differences. Samples with P values of <0.05 were considered statistically different.",
    "LLM": "MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The E. coli strains and plasmids used in this study are listed in Table 1. They were grown aerobically at 37\u00b0C in Luria-Bertani (LB) medium. Construction of azoR knockout mutant. The one-step replacement method described by Datsenko and Wanner (12) was used to construct an azoR deletion in E. coli JM109. PCR was done using pKD4 as a template and primers D1 (5-TGGACTGAATATCTACAGTCCACATCAAGACCGTGTCCGGCATA TGAATATCCTCCTTAG-3) and D2 (5-AACGGGGCATCGCCCATTCAA ACATCTATAAGGAAACACCGTGTAGGCTGGAGCTGCTTC-3). The un\ufffederlined sequences anneal to the template plasmid, while the remaining se\ufffequences correspond to the ends of azoR. The PCR-amplified DNA fragment was electroporated into E. coli ADH containing plasmid pKD46, which expresses Red recombinase and was cured later by growth at 37\u00b0C. The mutant strain E. coli LG1 was selected on an LB plate supplemented with kanamycin. The dele\ufffetion of the gene was confirmed by PCR and sequencing. Cloning of azoR from E. coli JM109. The azoR gene was PCR-amplified from the chromosomal DNA of E. coli JM109 by using primers P1 (5-GAATTCAT GAGCAAGGTATTAGTTCTTAA-3) and P2 (5-CTCGAGTTATGCAGAA ACAATGCTGTCG-3). The amplified DNA fragment was digested with EcoRI and XhoI and cloned into the EcoRI/XhoI double-digested pGEX-4T-1. The resulting plasmid pGEX-azoR was transformed into E. coli JM109, and the resultant E. coli LG2 was used for the expression of GSH S-transferase (GST) fusion AzoR protein. Purification and reductase activity assay of AzoR. E. coli LG2 cells were grown in LB medium to an optical density at 600 nm (OD600) of 0.5, and then the azoR gene was induced by adding 0.5 mM isopropyl--D-thiogalactopyranoside to the cultures and further incubating them for 3 additional hours. Cells were then harvested by centrifugation at 4,000 \u0004 g for 15 min and washed twice with 20 mM sodium phosphate buffer, pH 7.0. The pellets were frozen at \u000580\u00b0C overnight. The cells that were thawed on ice and resuspended in phosphate buffer were disrupted by ultrasonication three times for 20 s each time and spun at 50,000 \u0004 g for 20 min at 4\u00b0C. The supernatant fraction was saved. The GST-AzoR protein was purified by glutathione Sepharose 4B (Amersham Pharmacia Biotech) ac\ufffecording to the manufacturer\u2019s instructions. The GST tag was removed from AzoR by digestion with 10 U of thrombin per mg of fusion protein. It was mixed gently and incubated in cleavage buffer (20 mM Tris-HCl [pH 8.5], 120 mM NaCl, and 2.5 mM CaCl2) at room temperature (22 to 25\u00b0C) for 16 h. The thrombin-treated samples were purified again with glutathione Sepharose 4B according to the manufacturer\u2019s instructions. The progress of AzoR purification was monitored by 12% sodium dodecyl sulfate-polyacrylamide gel electrophore\ufffesis. The protein concentration was determined by the method of Bradford (5), with bovine serum albumin as a standard. The standard reductase activity assay system comprised 20 mM sodium phos\ufffephate buffer (pH 7.0), 10 to 700 \u0006M electron acceptor substrate (methyl red or various quinone compounds), 100 \u0006M NADH, 20 \u0006M FMN, and a suitable amount of enzyme. The reaction was initiated by the addition of NADH. Re\ufffeductase activities were monitored based on the oxidation of NADH at 340 nm (\u03b5 \u0007 6.22 mM\u00051 cm\u00051 ) (26). Values of Km (\u0006M) and kcat (s\u00051 ) were obtained from Lineweaver-Burk plots, and kcat/Km (s\u00051 mM\u00051 ) values were calculated. Effects of stressors on growth of E. coli strains. The overnight cultures of E. coli JM109 and E. coli LG1 were used to inoculate fresh LB medium to a starting OD600 of 0.1. They were grown at 37\u00b0C aerobically until the OD600 reached 0.6. Then each culture was split equally into several portions and treated with stres\ufffesors, including 0.5 mM 2-MHQ, 1 mM diamide, 6 mM catechol, 0.3 mM men\ufffeadione, and 1 and 10 mM H2O2. Intracellular GSH measurements were made using a GSH assay kit (Calbiochem, San Diego, CA). To study the effects of exogenous GSH on the growth of E. coli JM109 and E. coli LG1 in the presence of 2-MHQ, 0.25 to 2 mM GSH was added to the cultures before the addition of 0.5 mM 2-MHQ. Quantitative PCR analysis. E. coli JM109 culture was grown at 37\u00b0C aerobi\ufffecally, and samples were taken in different growth phases to study the transcrip\ufffetion of azoR under normal growth conditions. Effects of different stressors on the transcription of azoR were evaluated. Various stressors were applied to E. coli cultures when the OD600 reached 0.6. Samples were taken before (0 min) and 10 and 20 min after addition of stressors. Total RNA was obtained with RNAiso reagent (TaKaRa Dalian). DNA con\ufffetamination was eliminated by a DNase I treatment at 37\u00b0C for 1 h. RNA integrity was checked by agarose gel electrophoresis. The concentration and purity of extracted RNA were determined by measuring the A260 and A280. In total, 0.5 \u0006g of purified RNA was reverse transcribed (15 min at 37\u00b0C and 5 s at 85\u00b0C) by using PrimeScript real-time (RT) enzyme mix I (TaKaRa Dalian). For the reverse transcription, 50 \u0006M Oligo dT primer and 100 \u0006M random hexamers were used in each reaction. Quantitative RT-PCR was performed with a TaqMan assay in a Thermal Cycler Dice RT system (TaKaRa Dalian) using the following condi\ufffetions: 10 s at 95\u00b0C and 45 cycles of 5 s at 95\u00b0C and 30 s at 60\u00b0C. The primers used for amplification of azoR cDNA were azoR-F (5-GGGATCCACAAAGATGG ACCAAC-3) and azoR-R (5-GGCGAAGACAAATTTCACATCGG-3), and the TaqMan probe was azoR-P (5-[FAM]-ACCTGGTGACGCCGTATCTGT CCACG-[Eclipse]-3). E. coli 16S rRNA was chosen as an endogenous control for normalization of the cDNA loading in each PCR. Primers used for amplifi\ufffecation of 16S rRNA cDNA were 16S rRNA-F (5-CCATGAAGTTGGAATCG CTAG-3) and 16S rRNA-R (5-GCTTGACGGGCGGTGT-3), and the TaqMan probe was 16S rRNA-P (5-[FAM]-TACAAGGCCCGGGAACGTATT CACCG-[Eclipse]-3). The reaction mixtures (25 \u0006l) contained 12.5 \u0006l of Premix Ex Taq (2\u0004) (TaKaRa Dalian), 10 \u0006M each primer, 3 \u0006M TaqMan probe, and 2-\u0006l portions of different dilutions of the reverse transcription product. The standard curves for quantification were calculated by serial 10-fold dilutions of the standard cDNA transcript. Control reaction in which reverse transcriptase was omitted from the reaction mixture ensured that DNA products resulted from the amplification of cDNA rather than from DNA contamination. Determination of TSP. RNA was extracted from cells exposed to 2-MHQ for 10 min. Determination of the transcription start point (TSP) was performed TABLE 1. Strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source or reference E. coli strains JM109 endA1 recA1 gyrA96 thi-1 hsdR17 relA1 supE44 \b(lac-proAB) Promega D JM109 pKD46 This study LG1 JM109 azoR::kan Kmr This study LG2 JM109 pGEX-azoR This study Plasmids pGEX-4T-1 Overexpression vector with GST tag; Ampr Promega pGEX-azoR pGEX-4T-1 containing azoR fragment from E. coli JM109; Ampr This study pKD46 Red recombinase-expressing plasmid; Ampr 12 pKD4 FRT-flanked kanamycin cassette template 12 a Kmr , kanamycin resistance; Ampr , ampicillin resistance. VOL. 191, 2009 AzoR PROVIDES RESISTANCE TO THIOL-SPECIFIC STRESS 6395 using the 5-Full rapid amplification of cDNA ends (RACE) kit (TaKaRa Dalian) according to manufacturer\u2019s instructions. The adaptor-ligated cDNA was amplified by two sequential reactions using azoR outer primer (5-CATCG CTCGGACGCAGA-3) and azoR inner primer (5-CAACCAGTTCGCCATC CAGTA-3). After denaturation at 94\u00b0C for 3 min, the amplification (30 cycles) was 98\u00b0C for 15 s, 52\u00b0C for 30 s, and 72\u00b0C for 2 min, followed by one cycle of 72\u00b0C for 10 min. The PCR products were sequenced to recognize the relevant TSP. Sequence alignment and structure analysis. Multiple sequence alignment was conducted using Clustal X 1.8 (10) and visualized with Genedoc 2.6 (32). Three\ufffedimensional structure analysis was conducted using PyMol 0.99 (DeLano Scien\ufffetific, San Carlos, CA). Statistical analysis. Student\u2019s t test was employed to investigate statistical differences. Samples with P values of 0.05 were considered statistically different."
  },
  "35080802": {
    "bleu": 93.27595503543829,
    "meteor": 92.88973026668728,
    "rouge1_p": 0.9379689844922461,
    "rouge1_r": 0.9625256673511293,
    "rouge1_f": 0.9500886749429946,
    "rouge2_p": 0.9254254254254254,
    "rouge2_r": 0.9496661530559836,
    "rouge2_f": 0.9373891001267427,
    "rougeL_p": 0.9349674837418709,
    "rougeL_r": 0.9594455852156057,
    "rougeL_f": 0.9470483911831771,
    "cosine_similarity": 0.9146374886400562,
    "precision": 0.9135802469135802,
    "recall": 0.9487179487179487,
    "ner_f1": 0.9308176100628931,
    "ner_tp": 148,
    "ner_fp": 14,
    "ner_fn": 8,
    "bertscore_p": 0.9532318711280823,
    "bertscore_r": 0.9657720327377319,
    "bertscore_f1": 0.9596445560455322,
    "bertscore_scibert_error": "The expanded size of the tensor (2992) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2992].  Tensor sizes: [1, 512]",
    "len_gt": 11039,
    "len_jl": 11628,
    "GT": "Experimental Section General experimental methods: Chemicals and reagents were purchased from TCI and Sigma-Aldrich. The expression vector, pET16bP, was used for the cloning of formate dehydrogenase (FDH) and azoreductase (AzoRo).[11,51,52,53] All constructs were initially transformed to Escherichia coli DH5\u03b1 then the plasmids were isolated using NucleoSpin plasmid mini prep kit for plasmid DNA (Macherey-Nagel). Preculture for expression strains were done using lysogeny broth (LB) with 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract.[53] A final concentration of 100 \u03bcg/mL ampicillin was used for LB broth and agar plates. Cultures were incubated at 37\u00b0C for 24 h, 140 rpm. Plasmid construction and cloning of the fusion protein: The cloning of the codon-optimized azoRo (KT923288) was described in the study of Qi et al.[11] Meanwhile, the gene fdh was also codon optimized for protein expression. The codon optimized fdh (OL449253) was synthesized (Eurofins) in a pEX_K4 plasmid. The gene was obtained via restriction digestion of NdeI and NotI sites. Both the azoRo and fdh fragments were ligated into a pET16bP vector using the same restriction sites. E. coli DH5\u03b1 was transformed with the plasmids and the plasmids were then isolated using the GeneJET Plasmid Mini Prep Kit (ThermoFischer). The inserts were checked via PCR using pETcheck primers (Table 3). The construction of the FDH \ud835\udc36 23 \ud835\udc46 / \ud835\udc37 195 \ud835\udc44 \ud835\udc4c 196 \ud835\udc3b C23S/D195QY196H \u200b was done by using the pET16bP_FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b as a template for a PCR reaction, amplifying the gene of interest with the overlapping primers C and D (Table 3) to change Asp195 to Glu and Tyr196 to His, allowing the possibility to accept NADP+.[27] The plasmids, pET16bP_AzoRo, pET16bP_FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b and pET16bP_FDH \ud835\udc36 23 \ud835\udc46 / \ud835\udc37 195 \ud835\udc44 \ud835\udc4c 196 \ud835\udc3b C23S/D195QY196H \u200b , were used as a parental template for the construction of the fusion proteins. A total of four genes encoding the fusion proteins were cloned via Gibson assembly.[55] As linker, a sequence encoding for a His 10 10 \u200b -tag was placed between the genes of azoRo and fdh. For pET16bP_AzoRo+FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b and pET16bP_AzoRo+FDH \ud835\udc37 195 \ud835\udc44 \ud835\udc4c 196 \ud835\udc3b D195QY196H \u200b , pET16bP_AzoRo was linearized via PCR (Eppendorf) using primers E and F (Table 3) with 30 cycles and used as the vector. Each cycle consists of denaturation for 15 s at 95\u00b0C, annealing for 15 s at 60\u00b0C, and elongation for 1 min 45 s at 72\u00b0C. The inserts FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b and FDH \ud835\udc37 195 \ud835\udc44 \ud835\udc4c 196 \ud835\udc3b D195QY196H \u200b was amplified via PCR from pET16bP_FDH using primers H+J (Table 3) with the following conditions: denaturation for 15 s at 95\u00b0C, annealing for 15 s at 58\u00b0C, and elongation for 45 s at 72\u00b0C. The two PCR products were digested overnight with DpnI and ligated together using NEBuilder Assembly Mastermix (NEB). For pET16bP_FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b +AzoRo and pET16bP_FDH \ud835\udc37 195 \ud835\udc44 \ud835\udc4c 196 \ud835\udc3b D195QY196H \u200b +AzoRo, the PCR conditions were the same. However, pET16bP_FDH was used as a template and primers E+G were used for vector amplification while for the insert AzoRo, the pET16bP_AzoRo was used as a template and primers I+J were used for the insert amplification. E. coli DH5\u03b1 was transformed with the constructs and the plasmids were purified (ThermoFisher Genejet Plasmid Miniprep Kit). The plasmids were controlled via Sanger sequencing (Microsynth Seqlab) using the primer K or L to check if the proper inserts were incorporated. Gene expression and protein purification: Plasmids of the fusion protein constructs were transformed to different E. coli T7 expression systems such as BL21(DE3), SHuffle, and C41(DE3). Transformation was done through heat-shock method by subjecting the cells at 42\u00b0C for 1 min. The cells were placed on ice for 3 min and a 950 \u03bcL antibiotic-free LB broth was pipetted into the tube. The regeneration was done at 37\u00b0C for 1 h. The cells were then spun down at 4,700\u00d7g for 7 min (ThermoScientific Heraeus Fresco 17). The supernatant was discarded, and the pellets were resuspended with LB medium. A 50 \u03bcL aliquot was plated onto an LB agar plate with ampicillin (100 \u03bcg/mL) and the plates were incubated at 37\u00b0C for 24 h. Isolated colony from the LB agar plate was taken using a sterile inoculating loop and transferred to an LB medium to serve as a preculture. After 24 h, the precultures were transferred to a fresh expression medium. Different expression media such as LB, LBNB, LB with 10% glycerol, and LB with 1% ethanol were used to optimize the best conditions for the fusion proteins production. The cultures were grown at 37\u00b0C until an OD of 0.5\u20130.8 was obtained. The temperature was lowered to 20\u00b0C and a final concentration of 0.1 mM isopropyl-\u03b2-D-1-thiogalactopyranoside (IPTG) was added to the culture for induction. The cells were incubated overnight and were harvested the next day at 5,000\u00d7g for 30 min (Sorvall RC 5C Plus). Cell pellets were stored at \u221220\u00b0C for further use. For protein purification, the cell pellets were thawed and resuspended with 25 mM Tris-HCl buffer, pH 7.5. DNAse I was added to the solution. The cells were then lysed using an ultrasonic cell disruptor with an MS72 tip (Sonopuls, Bandelin, VWR). The conditions for the cell lysis were as follows: 30 s with 1 min rest, repeating the process 10 times. The disrupted cell cultures were centrifuged for 30 min at 17,000\u00d7g at 4\u00b0C (ThermoScientific Heraeus Fresco 17) to obtain the crude extract. The crude cell extracts were then subjected for protein purification through fast protein liquid chromatography (FPLC, \u00c4kta Start, GE Healthcare) using a nickel affinity column (HisTrap, GE Healthcare). Tris-HCl buffer (25 mM Tris, 500 mM NaCl, pH 7.5) was used as the binding buffer. The bounded fusion protein was eluted from the nickel column by employing a gradient of 20 to 100% Tris-HCl buffer containing imidazole (25 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5). The purified proteins were precipitated using a saturated ammonium sulfate solution. Pellets obtained were then re-dissolved with 100 mM phosphate buffer, pH 7. Different samples were taken during protein expression and purification such as after induction, crude extract, cell pellet, flowthrough, washing, and elution for sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS-PAGE) analysis.[55] This was to verify the fusion protein according to its size. The protein samples were mixed with a 2\u00d7 SDS sample buffer (300 mM Tris-HCl pH 6.8, 4% SDS, 30% glycerol, 5% \u03b2-mercaptoethanol, 0.2% dithiothreitol, and 0.01% bromophenol blue) and were denatured at 90\u00b0C for 10 mins. The protein samples were then pipetted into the wells of the SDS gel and the Thermo Scientific PageRuler prestained protein ladder was used as the marker. Proteins were quantified using the Bradford Assay (Rotiquant; Carl Roth, Germany) using bovine serum albumin as the standard.[56,57] Biochemical characterization of the fusion protein: The activities of the fusion protein were measured using a UV-vis spectrophotometer (Cary 60 UV-vis Agilent) at 25\u00b0C. FDH part of FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b +AzoRo was measured in a quartz cuvette containing 1 mM NAD+, 125 mM formate, and the enzyme. NADH generation at 340 nm was followed. Meanwhile, azoreductase part of FDH \ud835\udc36 23 \ud835\udc46 C23S \u200b +AzoRo was also carried out in a quartz cuvette containing 150 \u03bcM NADH, 50 \u03bcM FMN, 25 \u03bcM of the azo dye substrate, and the enzyme. Degradation of azo dyes at their respective wavelengths were followed ((1) 430 nm and (5) 571 nm). The optimum pH for each enzyme of the fusion protein was determined by checking activities for each segment at pH 6, 7, and 8. For thermal stability, the proteins were incubated at different time points and the enzyme activity was tested for each time point. For metal ions, the components for a cuvette reaction were the same but just supplemented with additional metal ions (Cu 2 + 2+ , Mg 2 + 2+ , Ca 2 + 2+ , Mn 2 + 2+ , Zn 2 + 2+ ) with a final concentration of 50 \u03bcM. Kinetic analysis of the fusion protein: Steady-state kinetic measurements were done as described above. Initial velocities of the enzymatic reaction for FDH and AzoRo part were measured. Apparent kinetic parameters were obtained through a non-linear Michael-Menten assumption. The specific activity was determined as described previously wherein 1 U represents the conversion of 1 \u03bcmol NAD+, 1 \u03bcmol NADH or substrate per min. For all enzymatic reactions, a total of 2.6 \u03bcg of proteins was used. In vitro degradation of azo dyes and assessment of substrate scope via HPLC: To further prove that the fusion protein was functional, in vitro degradation of dyes was conduction. The components of the biocatalytic reactions comprised of 125 mM formate, 1 mM NAD+, 50 \u03bcM FMN, 100 \u03bcM of the substrate, and 2.6 \u03bcg enzyme. The reactions were done on a 1.5 mL microcentrifuge tube at 30\u00b0C with a shaking of 600 rpm (LLG-uniTHERMIX 2 pro). A 50 \u03bcL volume was obtained from the samples and the reaction was stopped with equal parts of methanol (MeOH). The mixture was then centrifuged for 30 min and the supernatant was carefully taken. The reaction sampling time points were done after 1 h, 20 h, 24 h, 40 h. Additional setup without the enzymes served as a negative control. All measurements were done in triplicates. The samples were subjected to reverse phase \u2013 high pressure liquid chromatography (RP-HPLC) analysis (ThermoScientific Dionex Ultimate 3000) to determine if the azo dyes were really degraded and that the results observed were not just because of pH change. A C18 reversed-phase column (Knauer Eurospher 100-5 C18, 125\u00d74 mm) was used. Different instrument methods were employed to elute the azo dyes properly. For (1), the dye was eluted using a 40\u201395% gradient of MeOH with 0.1% TFA H 2 2 \u200b O at a flow rate of 0.7 mL/min, detecting it at 490 nm. For (5), an isocratic method of 50% MeOH and 50% H 2 2 \u200b O with 50 mM ammonium acetate was used at a flow rate of 1.0 mL/min, via 575 nm. (2) was also eluted using an isocratic method composed of 60% MeOH and 40% H 2 2 \u200b O with 0.1% TFA at a flow rate of 0.7 mL/min, detecting it at 440 nm. For (6) and (4), 520 nm and 508 nm were used to detect the dyes, respectively, with a buffer composition of 70% MeOH and 30% ammonium acetate at 0.7 mL/min. For (3), 80% MeOH and 20% H 2 2 \u200b O with 0.1% TFA was used at a flow rate of 0.7 mL/min, detecting it at 375 nm. Extraction and analysis of dye degradation product: LC-MS analysis of the decolorization products were performed with a LC-MS 8030 (Shimadzu). Separation was conducted through a reverse phase C18 column (Kinetex\u00ae, 2.6 \u03bcm, 100 \u00c5, 100\u00d72.1 mm) and a guard column at 40\u00b0C with mobile phase A, consisting of water with 10 mM ammonium acetate (LiChropur\u2122, Merck, Germany) and mobile phase B, consisting of methanol. Elution program was 10% B for the first 2 min, followed by a linear gradient up to 90% B for 6 min, at a flow rate of 0.4 mL/min. Products were detected by a diode array detector (DAD) at 254 nm. Mass spectrometry was performed with an electron spray ionization (ESI) unit in negative mode.",
    "LLM": "Experimental Section General experimental methods: Chemicals and reagents were purchased from TCI and Sigma-Aldrich. The expression vector, pET16bP, was used for the cloning of formate dehydrogenase (FDH) and azoreductase (AzoRo).[11,51,52,53] All constructs were initially transformed to Escherichia coli DH5\u03b1 then the plasmids were isolated using NucleoSpin plasmid mini prep kit for plasmid DNA (Macherey-Nagel). Preculture for expression strains were done using lysogeny broth (LB) with 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract.[53] A final concentration of 100 \u03bcg/mL ampicillin was used for LB broth and agar plates. Cultures were incubated at 37\u00b0C for 24 h, 140 rpm. Plasmid construction and cloning of the fusion protein: The cloning of the codon-optimized azoRo (KT923288) was described in the study of Qi et al.[11] Meanwhile, the gene fdh was also codon optimized for protein expression. The codon optimized fdh (OL449253) was synthesized (Eurofins) in a pEX_K4 plasmid. The gene was obtained via restriction digestion of NdeI and NotI sites. Both the azoRo and fdh fragments were ligated into a pET16bP vector using the same restriction sites. E. coli DH5\u03b1 was transformed with the plasmids and the plasmids were then isolated using the GeneJET Plasmid Mini Prep Kit (ThermoFischer). The inserts were checked via PCR using pETcheck primers (Table 3). The construction of the FDHC23SD195QY196H was done by using the pET16bP_FDHC23S as a template for a PCR reaction, amplifying the gene of interest with the overlapping primers C and D (Table 3) to change Asp195 to Glu and Tyr196 to His, allowing the possibility to accept NADP+.[27] The plasmids, pET16bP_AzoRo, pET16bP_FDHC23S and pET16bP_ FDHC23SD195QY196H, were used as a parental template for the construction of the fusion proteins. A total of four genes encoding the fusion proteins were cloned via Gibson assembly.[55] As linker, a sequence encoding for a His10-tag was placed between the genes of azoRo and fdh. For pET16bP_AzoRo+FDHC23S and pET16bP_AzoRo+FDHD195QY196H, pET16bP_AzoRo was linearized via PCR (Eppendorf) using primers E and F (Table 3) with 30 cycles and used as the vector. Each cycle consists of denaturation for 15 s at 95\u00b0C, annealing for 15 s at 60\u00b0C, and elongation for 1 min 45 s at 72\u00b0C. The inserts FDHC23S and FDHD195QY196H was amplified via PCR from pET16bP_FDH using primers H+J (Table 3) with the following conditions: denaturation for 15 s at 95\u00b0C, annealing for 15 s at 58\u00b0C, and elongation for 45 s at 72\u00b0C. The two PCR products were digested overnight with DpnI and ligated together using NEBuilder Assembly Mastermix (NEB). For pET16bP_FDHC23S+AzoRo and pET16bP_FDHD195QY196H+AzoRo, the PCR conditions were the same. However, pET16bP_FDH was used as a template and primers E+G were used for vector amplification while for the insert AzoRo, the pET16bP_AzoRo was used as a template and primers I+J were used for the insert amplification. E. coli DH5\u03b1 was transformed with the constructs and the plasmids were purified (ThermoFisher Genejet Plasmid Miniprep Kit). The plasmids were controlled via Sanger sequencing (Microsynth Seqlab) using the primer K or L to check if the proper inserts were incorporated. Gene expression and protein purification: Plasmids of the fusion protein constructs were transformed to different E. coli T7 expression systems such as BL21(DE3), SHuffle, and C41(DE3). Transformation was done through heat-shock method by subjecting the cells at 42\u00b0C for 1 min. The cells were placed on ice for 3 min and a 950 \u03bcL antibiotic-free LB broth was pipetted into the tube. The regeneration was done at 37\u00b0C for 1 h. The cells were then spun down at 4,700\u00d7g for 7 min (ThermoScientific Heraeus Fresco 17). The supernatant was discarded, and the pellets were resuspended with LB medium. A 50 \u03bcL aliquot was plated onto an LB agar plate with ampicillin (100 \u03bcg/mL) and the plates were incubated at 37\u00b0C for 24 h. Isolated colony from the LB agar plate was taken using a sterile inoculating loop and transferred to an LB medium to serve as a preculture. After 24 h, the precultures were transferred to a fresh expression medium. Different expression media such as LB, LBNB, LB with 10% glycerol, and LB with 1% ethanol were used to optimize the best conditions for the fusion proteins production. The cultures were grown at 37\u00b0C until an OD of 0.5\u20130.8 was obtained. The temperature was lowered to 20\u00b0C and a final concentration of 0.1 mM isopropyl-\u03b2-D-1-thiogalactopyranoside (IPTG) was added to the culture for induction. The cells were incubated overnight and were harvested the next day at 5,000\u00d7g for 30 min (Sorvall RC 5C Plus). Cell pellets were stored at \ufffe 20\u00b0C for further use. For protein purification, the cell pellets were thawed and resuspended with 25 mM Tris-HCl buffer, pH 7.5. DNAse I was added to the solution. The cells were then lysed using an ultrasonic cell disruptor with an MS72 tip (Sonopuls, Bandelin, VWR). The conditions for the cell lysis were as follows: 30 s with 1 min rest, repeating the process 10 times. The disrupted cell cultures were centrifuged for 30 min at 17,000\u00d7g at 4\u00b0C (ThermoScientific Heraeus Fresco 17) to obtain the crude extract. The crude cell extracts were then subjected for protein purification through fast protein liquid chromatography (FPLC, \u00c4kta Start, GE Healthcare) using a nickel affinity column (HisTrap, GE Healthcare). Tris-HCl buffer (25 mM Tris, 500 mM NaCl, pH 7.5) was used as the binding buffer. The bounded fusion protein was eluted from the nickel column by employing a gradient of 20 to 100% Tris-HCl buffer containing imidazole (25 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5). The purified proteins were precipitated using a saturated ammonium sulfate solution. Pellets obtained were then re-dissolved with 100 mM phosphate buffer, pH 7. Different samples were taken during protein expression and purification such as after induction, crude extract, cell pellet, flowthrough, washing, and elution for sodium dodecyl sulfate\ufffeTable 3. Primers used in the study. Each primer is designated with a letter. Primer name Primer sequence PET_check fw 5\u2019-CATCACAGCAGCGGCCATATCGAAG-3\u2019 A PET_check rv 5\u2019-CAGCTTCTTTTCGGGCTTTGTTAG-3\u2019 B FDH_ D195QY196H_ fw 5\u2019-TTAACCCGAAAGAACTGCTGTATTACCAG CATCAGGCATTACCGAAAGAAGCCG-3\u2019 C FDH_ D195QY196H_rv 5\u2019-CGGCTTCTTTCGGTAATGCCTGATGC TGGTAATACAGCAGTTCTTTCGGGTTAA-3\u2019 D PET-fw-Gibson 5\u2019-TGAGCGGCCGCACTTAAGTTACG CGTGGATCC-3\u2019 E PET-Azorev-Gibson 5\u2019-ACGTACAAGAGAAGTCACC CAACGTTGGTC-3\u2019 F PET-FDHrev-Gibson 5\u2019-TTTCTTATCGTGTTTCCCA TACGCTTTCG-3\u2019 G Overlap-AzoFDH\ufffeGibson 5\u2019-GGGTGACTTCTCTTGTACGTGGCCATCAT CAT CATCATCATC-3\u2019 H Overlap-FDHAzo\ufffeGibson 5\u2019-ATGGGAAACACGATAAGAAAGGCCATCAT CATCATCATCATC-3\u2019 I Overlap-insert-PET 5\u2019-GTAACTTAAGTGCGGCCGCTCA-3\u2019 J AzoRo_ internalcheck 5\u2019-GAGCACCTGGTTCCATTAGC-3\u2019 K FDH_ internalcheck 5\u2019-GGCAAATTTGATTATCGCCCAC-3\u2019 L polyacrylamide gel electrophoresis (SDS-PAGE) analysis.[55] This was to verify the fusion protein according to its size. The protein samples were mixed with a 2\u00d7 SDS sample buffer (300 mM Tris-HCl pH 6.8, 4% SDS, 30% glycerol, 5% \u03b2-mercaptoethanol, 0.2% dithiothreitol, and 0.01% bromophenol blue) and were denatured at 90\u00b0C for 10 mins. The protein samples were then pipetted into the wells of the SDS gel and the Thermo Scientific PageRuler prestained protein ladder was used as the marker. Proteins were quantified using the Bradford Assay (Rotiquant; Carl Roth, Germany) using bovine serum albumin as the standard.[56,57] Biochemical characterization of the fusion protein: The activities of the fusion protein were measured using a UV-vis spectrophotometer (Cary 60 UV-vis Agilent) at 25\u00b0C. FDH part of FDHC23S+ AzoRo was measured in a quartz cuvette containing 1 mM NAD+, 125 mM formate, and the enzyme. NADH generation at 340 nm was followed. Meanwhile, azoreductase part of FDHC23S+AzoRo was also carried out in a quartz cuvette containing 150 \u03bcM NADH, 50 \u03bcM FMN, 25 \u03bcM of the azo dye substrate, and the enzyme. Degradation of azo dyes at their respective wavelengths were followed ((1) 430 nm and (5) 571 nm). The optimum pH for each enzyme of the fusion protein was determined by checking activities for each segment at pH 6, 7, and 8. For thermal stability, the proteins were incubated at different time points and the enzyme activity was tested for each time point. For metal ions, the components for a cuvette reaction were the same but just supplemented with additional metal ions (Cu2+, Mg2+, Ca2+, Mn2+, Zn2+) with a final concentration of 50 \u03bcM. Kinetic analysis of the fusion protein: Steady-state kinetic measurements were done as described above. Initial velocities of the enzymatic reaction for FDH and AzoRo part were measured. Apparent kinetic parameters were obtained through a non-linear Michael-Menten assumption. The specific activity was determined as described previously wherein 1 U represents the conversion of 1 \u03bcmol NAD+, 1 \u03bcmol NADH or substrate per min. For all enzymatic reactions, a total of 2.6 \u03bcg of proteins was used. In vitro degradation of azo dyes and assessment of substrate scope via HPLC: To further prove that the fusion protein was functional, in vitro degradation of dyes was conduction. The components of the biocatalytic reactions comprised of 125 mM formate, 1 mM NAD+, 50 \u03bcM FMN, 100 \u03bcM of the substrate, and 2.6 \u03bcg enzyme. The reactions were done on a 1.5 mL microcentrifuge tube at 30\u00b0C with a shaking of 600 rpm (LLG\ufffeuniTHERMIX 2 pro). A 50 \u03bcL volume was obtained from the samples and the reaction was stopped with equal parts of methanol (MeOH). The mixture was then centrifuged for 30 min and the supernatant was carefully taken. The reaction sampling time points were done after 1 h, 20 h, 24 h, 40 h. Additional setup without the enzymes served as a negative control. All measurements were done in triplicates. The samples were subjected to reverse phase \u2013 high pressure liquid chromatography (RP-HPLC) analysis (ThermoScientific Dionex Ultimate 3000) to determine if the azo dyes were really degraded and that the results observed were not just because of pH change. A C18 reversed-phase column (Knauer Eurospher 100-5 C18, 125\u00d7 4 mm) was used. Different instrument methods were employed to elute the azo dyes properly. For (1), the dye was eluted using a 40\u2013 95% gradient of MeOH with 0.1% TFA H2O at a flow rate of 0.7 mL/ min, detecting it at 490 nm. For (5), an isocratic method of 50% MeOH and 50% H2O with 50 mM ammonium acetate was used at a flow rate of 1.0 mL/min, via 575 nm. (2) was also eluted using an isocratic method composed of 60% MeOH and 40% H2O with 0.1% TFA at a flow rate of 0.7 mL/min, detecting it at 440 nm. For (6) and (4), 520 nm and 508 nm were used to detect the dyes, respectively, with a buffer composition of 70% MeOH and 30% ammonium acetate at 0.7 mL/min. For (3), 80% MeOH and 20% H2O with 0.1% TFA was used at a flow rate of 0.7 mL/min, detecting it at 375 nm. Extraction and analysis of dye degradation product: LC-MS analysis of the decolorization products were performed with a LC\ufffeMS 8030 (Shimadzu). Separation was conducted through a reverse phase C18 column (Kinetex\u00ae, 2.6 \u03bcm, 100 \u00c5, 100\u00d72.1 mm) and a guard column at 40\u00b0C with mobile phase A, consisting of water with 10 mM ammonium acetate (LiChropur\u2122, Merck, Germany) and mobile phase B, consisting of methanol. Elution program was 10% B for the first 2 min, followed by a linear gradient up to 90% B for 6 min, at a flow rate of 0.4 mL/min. Products were detected by a diode array detector (DAD) at 254 nm. Mass spectrometry was performed with an electron spray ionization (ESI) unit in negative mode."
  },
  "11583992": {
    "bleu": 97.34713031361663,
    "meteor": 97.2744650512963,
    "rouge1_p": 0.9907786885245902,
    "rouge1_r": 0.9907786885245902,
    "rouge1_f": 0.9907786885245902,
    "rouge2_p": 0.9856483854433624,
    "rouge2_r": 0.9856483854433624,
    "rouge2_f": 0.9856483854433624,
    "rougeL_p": 0.9907786885245902,
    "rougeL_r": 0.9907786885245902,
    "rougeL_f": 0.9907786885245902,
    "cosine_similarity": 0.9642174976201504,
    "precision": 0.9041916167664671,
    "recall": 0.9151515151515152,
    "ner_f1": 0.9096385542168675,
    "ner_tp": 151,
    "ner_fp": 16,
    "ner_fn": 14,
    "bertscore_p": 0.9837315678596497,
    "bertscore_r": 0.9877527952194214,
    "bertscore_f1": 0.9857971668243408,
    "bertscore_scibert_error": "The expanded size of the tensor (2789) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2789].  Tensor sizes: [1, 512]",
    "len_gt": 10929,
    "len_jl": 10920,
    "GT": "EXPERIMENTAL PROCEDURES Materials E. coli JM109, Q-Sepharose FF, Q-Sepharose HP, Blue-Sepharose, Sephacryl S200, phenyl-Sepharose, and a Superdex 200 HR 10/30 pre\u00adpack column were from Amersham Pharmacia Biotech. GIGAPITE was from Seikagaku (Tokyo, Japan). Methyl red, FAD, FMN, 2-aminoben\u00adzoic acid (ABA), and N,N-dimethyl-p-phenylenediamine (DMPD) were from Nacalai Tesque (Kyoto, Japan). Ethyl red (4-diethylaminoazoben\u00adzene-2-carboxylic acid) and Ponceau SX (3-[(2,4-dimethyl-5-sulfopheny\u00adl)azo]-4-hydroxy-1-naphthalenesulfonic acid) were from Tokyo Kasei (Tokyo, Japan). Menadione was from Sigma. NAD(P)H were from Ori\u00adental Yeast (Tokyo, Japan). Assaying of Azoreductase The standard assay system for azoreductase comprised 25 mM Tris\u00adHCl (pH 7.4), 25 \u00b5M methyl red, 0.1 mM NADH, 20 \u00b5M FMN, and enzyme, in a final volume of 2 ml. The reaction was initiated by addition of the enzyme. The initial reaction rate was determined by monitoring the decrease in absorbance at 430 nm in the first 1.5 min in a glass cuvette of 1.0-cm light path at 30 \u00b0C. The enzyme activity was a linear function of the incubation time. One unit of methyl red reductase activity was defined as the amount catalyzing the degradation of 1 \u00b5mol of methyl red/min at 30 \u00b0C using a molar absorption coefficient of 23,360 M\u207b\u00b9 cm\u207b\u00b9. When methyl red was replaced with other azo dyes, the following molar absorption coefficients and wavelengths were used: 21,560 M\u207b\u00b9 cm\u207b\u00b9 (ethyl red at 450 nm) and 18,680 M\u207b\u00b9 cm\u207b\u00b9 (Ponceau SX at 500 nm). We confirmed that the absorption of NADH did not affect the absorption of the azo dyes. One unit of menadione reductase activity was defined as the amount catalyzing the reduction of 1 \u00b5mol NAD(P)H/min at 30 \u00b0C using a molar absorption coefficient of 6220 M\u207b\u00b9 cm\u207b\u00b9. Purification of Azoreductase All procedures were carried out at 4 \u00b0C. Preparation of Cell Extracts\u2014E. coli (JM109) cells that had been grown at 37 \u00b0C as 10-liter cultures in LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, pH 7.0) were resuspended in 160 ml of buffer A (20 mM Tris-HCl (pH 8.0), 1 mM EDTA). Lysozyme was added to give a final concentration of 0.1 mg/ml, and then the solution was incubated at 30 \u00b0C for 20 min. The viscous solution was chilled and subjected to sonication disruption (180-watt output, 30 s three times). Cell debris was removed by centrifugation at 12,500 \u00d7 g for 15 min. Ion Exchange Chromatography on Q-Sepharose FF\u2014The cell extract was applied at 0.6 ml/min to a Q-Sepharose FF column (16 \u00d7 2.5 cm) that had been pre-equilibrated with buffer A. After washing of the column with 800 ml of buffer A at 0.6 ml/min, azoreductase was eluted with 400 ml of buffer A containing a linear gradient of NaCl, from 0 to 0.6 M. Each fraction (fraction size, 6 ml) was subjected to activity measurement; the seven fractions containing the highest levels of activity were pooled. Affinity Chromatography on Blue-Sepharose\u2014The pooled fraction was applied at 0.5 ml/min to a Blue-Sepharose column (6.5 \u00d7 2.0 cm) that had been pre-equilibrated with buffer A. After washing of the column with 200 ml of buffer A, azoreductase was eluted with 70 ml of buffer A containing 0.4 mM NADH. Gel Filtration Chromatography on Sephacryl S200\u2014The eluate was concentrated to 6 ml on a YM-10 membrane and then applied at 0.3 ml/min to a Sephacryl S200 column (90 \u00d7 1.6 cm) that had been pre-equilibrated with buffer B (20 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 2 mM 2-mercaptoethanol). The two fractions (fraction size, 3 ml) contain\u00ading the highest activity were pooled. Hydroxyapatite Chromatography on GIGAPITE\u2014The pooled frac\u00adtion was applied at 0.3 ml/min to a GIGAPITE column (3 \u00d7 1.4 cm) that had been pre-equilibrated with buffer B. After washing of the column with buffer B at 0.3 ml/min, azoreductase was eluted with 60 ml of buffer B containing a linear gradient of potassium phosphate (pH 7.5), from 0 to 50 mM. The 10 fractions (fraction size, 3 ml) containing activity were pooled, and the buffer was exchanged with buffer A. Ion Exchange Chromatography on Q-Sepharose HP\u2014The enzyme solution was applied at 0.3 ml/min to a Q-Sepharose HP column (1.5 \u00d7 1.4 cm) that had been pre-equilibrated with buffer A. After washing of the column with buffer A, azoreductase was eluted with 20 ml of buffer A containing a linear gradient of NaCl, from 0 to 0.6 M. The 10 fractions (fraction size, 2 ml) containing activity were pooled. Hydrophobic Interaction Chromatography on Phenyl-Sepharose\u2014 Ammonium sulfate was dissolved in the pooled fraction to obtain 30% saturation (164 g/liter). The solution was applied at 0.3 ml/min to a phenyl-Sepharose column (1 by 0.8 cm) that had been pre-equilibrated with buffer A containing ammonium sulfate (30% saturation). The column was washed with the buffer, and then 40 ml of a linear gradient of ammonium sulfate, 30\u20130% saturation, in buffer A was applied. The fraction containing activity was concentrated and stored at \u221220 \u00b0C after adding glycerol to give 20% (v/v) concentration. N-terminal Amino Acid Sequencing One microgram of the purified enzyme was run on a 12.5% SDS\u00adpolyacrylamide Tricine gel (1 mm thick) (17), and then blotted onto a ProBlot\u00ae poly(vinylidene difluoride) membrane (Applied Biosystems). The protein band was examined with an Applied Biosystems model 477A protein sequencer, fitted with an on-line model 120A analyzer for the detection of phenylthiohydantoin-amino acids. Expression and Purification of the acpD Gene Product The acpD gene was obtained by PCR using genomic DNA of E. coli strain JM109 as the template. Pfu turbo DNA polymerase (Stratagene) and oligonucleotide primers (sense, 5-cggccatatgagcaaggtattagtt-3 containing an NdeI site; antisense, 5-gcgctcgagttatgcagaaacaat-3 con\u00adtaining a XhoI site) were used for PCR. The resulting DNA was sub\u00adcloned into the corresponding restriction site of pET22b to obtain a plasmid designated as pETacpD. The nucleotide sequence of the cloned DNA was confirmed by dideoxy sequencing. E. coli JM109 (DE3) har\u00adboring pETacpD was grown at 37 \u00b0C in LB medium. Expression was induced with 0.5 mM isopropyl-1-thio-\u03b2-D-galactopyranoside (IPTG) for 3 h with an optical density at 600 nm of 0.6. The recombinant AcpD was purified by Q-Sepharose, Blue-Sepharose, Sephacryl S200, and GIGA\u00adPITE column chromatographies. Stoichiometry of Azo Reduction Using the standard assay system (2 ml) with 7.8 ng of the purified acpD gene product (designated as AcpD), the methyl red degradation rate and NADH consumption rate in the first 1.5 min were determined by monitoring the absorbance at 430 and 340 nm, respectively. Because the absorbance of methyl red at 340 nm was a linear function of the absorbance at 430 nm under the given conditions, the absorbance at 340 nm in the standard assay system was corrected by calculation. The molar absorption coefficients were as described above. Identification of Reaction Products Methyl red was incubated in 40 ml of the standard assay system for 10 min at 30 \u00b0C with the purified AcpD (20 \u00b5g). For extraction of ABA, an aliquot (20 ml) of the reaction mixture was extracted with an equal volume of ethyl acetate three times, subsequent to adjusting the pH to 3 with 1 M HCl. The extracts were pooled and evaporated in a rotary evaporator. For DMPD, another aliquot (20 ml) was extracted with an equal volume of n-hexane three times, subsequent to adjusting the pH to 10 with 1 M NaOH. The extracts were pooled and evaporated in a rotary evaporator. Each residue was dissolved in 0.5 ml of acetonitrile. Each sample was diluted 200-fold with acetonitrile and then analyzed (20 \u00b5l) with a Shimadzu HPLC system equipped with a model SPD-6AV variable wavelength detector (detection wavelength: 336 nm for ABA; 250 nm for DMPD) and a Jasco CrestPak ODS column (4.6 mm \u00d7 150 mm). The mobile phase was composed of 25 mM phosphate and aceto\u00adnitrile, 5:95 (25 mM phosphate:acetonitrile). The flow rate was 0.8 ml/min. Overexpression and Purification of E. coli ACP The gene for ACP was amplified using genomic DNA of E. coli strain JM109 as the template for PCR. The forward primer included an EcoRI site before the start codon: 5-ctggaattcatgagcactatcga-3. The reverse primer included a HindIII restriction site after the stop codon: 5-ctgaagcttttacgcctggtggc-3. The PCR product was subcloned into the EcoRI/HindIII site of the pKK223\u20133 plasmid, and the resulting plasmid was designated as pKKacpP. E. coli JM109 was transformed with pKKacpP and grown at 37 \u00b0C with LB medium. After the cultures had been grown to an optical density at 600 nm of 0.6, expression was induced with 0.5 mM IPTG for 3 h. The purification procedure followed the method of Therisod et al. (18). In Vitro Assay for ACP Phosphodiesterase Activity The assay system comprised 50 mM Tris-HCl (pH 8.5), 0.02 mM MnCl\u2082, 25 mM MgCl\u2082, 1 mM dithiothreitol, 20 \u00b5g of holo/apoACP, and 3.7 \u00b5g of purified AcpD, in a final volume of 0.1 ml. After incubation at 35 \u00b0C for 12 h, a sample was analyzed by native PAGE (19). For the holoACP standard, 20 \u00b5g of holo/apoACP was converted to holoACP under the conditions with 50 mM Tris-HCl (pH 8.8), 0.1 mM CoA, 25 mM MgCl\u2082, 1 mM DTT, and 4.2 \u00b5g of ACP synthase. ACP synthase was expressed and purified by a procedure based on that of Lambalot and Walsh (20). For the apoACP standard, Ser36-substituted ACP was used. PCR-based site-directed mutagenesis was carried out with substitution from Ser to Cys. The expression and purification procedure were the same as those for the wild type. In Vivo Assaying of ACP Phosphodiesterase Activity ACP was coexpressed with AcpD in E. coli and then analyzed by native PAGE. To obtain a plasmid (designated as pACYCacpP) that was compatible with pETacpD and produced ACP, the BamHI-HincII 0.6-kb DNA fragment of pACYC177 (21) was replaced by the 1.4-kb BamHI\u00adPvuI fragment (the cohesive end of the PvuI site was blunt-ended) from pKKacpP. The 1.4-kb fragment contained a tac promoter, the ACP gene, and rrnB ribosomal RNA transcription terminators. E. coli JM109 (DE3) was cotransformed with pACYCacpP and pETacpD or pET21a (for a control experiment). Expression was induced by the addition of IPTG as described above. Other Techniques Protein concentrations were determined using protein assay reagent (Bio-Rad), with bovine serum albumin as the standard. SDS-PAGE was carried out using 12.5% gels as described by Laemmli (22). The proteins on gels were stained using a Silver Stain Kit II (Wako Pure Chemicals, Osaka, Japan). The native molecular weight of the protein was determined by gel filtration on a Superdex 200 HR column (30 \u00d7 1.0 cm) that had been equilibrated with Tris-buffered saline buffer (20 mM Tris-HCl (pH 7.5) and 0.15 M NaCl). Calibration of the column was carried out with the following proteins: alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa), all from Sigma.",
    "LLM": "EXPERIMENTAL PROCEDURES Materials E. coli JM109, Q-Sepharose FF, Q-Sepharose HP, Blue-Sepharose, Sephacryl S200, phenyl-Sepharose, and a Superdex 200 HR 10/30 pre\ufffepack column were from Amersham Pharmacia Biotech. GIGAPITE was from Seikagaku (Tokyo, Japan). Methyl red, FAD, FMN, 2-aminoben\ufffezoic acid (ABA), and N,N-dimethyl-p-phenylenediamine (DMPD) were from Nacalai Tesque (Kyoto, Japan). Ethyl red (4-diethylaminoazoben\ufffezene-2-carboxylic acid) and Ponceau SX (3-[(2,4-dimethyl-5-sulfopheny\ufffel)azo]-4-hydroxy-1-naphthalenesulfonic acid) were from Tokyo Kasei (Tokyo, Japan). Menadione was from Sigma. NAD(P)H were from Ori\ufffeental Yeast (Tokyo, Japan). Assaying of Azoreductase The standard assay system for azoreductase comprised 25 mM Tris\ufffeHCl (pH 7.4), 25 \u0001M methyl red, 0.1 mM NADH, 20 \u0001M FMN, and enzyme, in a final volume of 2 ml. The reaction was initiated by addition of the enzyme. The initial reaction rate was determined by monitoring the decrease in absorbance at 430 nm in the first 1.5 min in a glass cuvette of 1.0-cm light path at 30 \u00b0C. The enzyme activity was a linear function of the incubation time. One unit of methyl red reductase activity was defined as the amount catalyzing the degradation of 1 \u0001mol of methyl red/min at 30 \u00b0C using a molar absorption coefficient of 23,360 M1 cm1 . When methyl red was replaced with other azo dyes, the following molar absorption coefficients and wavelengths were used: 21,560 M1 cm1 (ethyl red at 450 nm) and 18,680 M1 cm1 (Ponceau SX at 500 nm). We confirmed that the absorption of NADH did not affect the absorption of the azo dyes. One unit of menadione reductase activity was defined as the amount catalyzing the reduction of 1 \u0001mol NAD(P)H/min at 30 \u00b0C using a molar absorption coefficient of 6220 M1 cm1 . Purification of Azoreductase All procedures were carried out at 4 \u00b0C. Preparation of Cell Extracts\u2014E. coli (JM109) cells that had been grown at 37 \u00b0C as 10-liter cultures in LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, pH 7.0) were resuspended in 160 ml of buffer A (20 mM Tris-HCl (pH 8.0), 1 mM EDTA). Lysozyme was added to give a final concentration of 0.1 mg/ml, and then the solution was incubated at 30 \u00b0C for 20 min. The viscous solution was chilled and subjected to sonication disruption (180-watt output, 30 s three times). Cell debris was removed by centrifugation at 12,500 \u0004 g for 15 min. Ion Exchange Chromatography on Q-Sepharose FF\u2014The cell extract was applied at 0.6 ml/min to a Q-Sepharose FF column (16 \u0004 2.5 cm) that had been pre-equilibrated with buffer A. After washing of the column with 800 ml of buffer A at 0.6 ml/min, azoreductase was eluted with 400 ml of buffer A containing a linear gradient of NaCl, from 0 to 0.6 M. Each fraction (fraction size, 6 ml) was subjected to activity measurement; the seven fractions containing the highest levels of ac\ufffetivity were pooled. Affinity Chromatography on Blue-Sepharose\u2014The pooled fraction was applied at 0.5 ml/min to a Blue-Sepharose column (6.5 \u0004 2.0 cm) that had been pre-equilibrated with buffer A. After washing of the column with 200 ml of buffer A, azoreductase was eluted with 70 ml of buffer A containing 0.4 mM NADH. Gel Filtration Chromatography on Sephacryl S200\u2014The eluate was concentrated to 6 ml on a YM-10 membrane and then applied at 0.3 ml/min to a Sephacryl S200 column (90 \u0004 1.6 cm) that had been pre-equilibrated with buffer B (20 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 2 mM 2-mercaptoethanol). The two fractions (fraction size, 3 ml) contain\ufffeing the highest activity were pooled. Hydroxyapatite Chromatography on GIGAPITE\u2014The pooled frac\ufffetion was applied at 0.3 ml/min to a GIGAPITE column (3 \u0004 1.4 cm) that had been pre-equilibrated with buffer B. After washing of the column with buffer B at 0.3 ml/min, azoreductase was eluted with 60 ml of buffer B containing a linear gradient of potassium phosphate (pH 7.5), from 0 to 50 mM. The 10 fractions (fraction size, 3 ml) containing activity were pooled, and the buffer was exchanged with buffer A. Ion Exchange Chromatography on Q-Sepharose HP\u2014The enzyme solution was applied at 0.3 ml/min to a Q-Sepharose HP column (1.5 \u0004 1.4 cm) that had been pre-equilibrated with buffer A. After washing of the column with buffer A, azoreductase was eluted with 20 ml of buffer A containing a linear gradient of NaCl, from 0 to 0.6 M. The 10 fractions (fraction size, 2 ml) containing activity were pooled. Hydrophobic Interaction Chromatography on Phenyl-Sepharose\u2014 Ammonium sulfate was dissolved in the pooled fraction to obtain 30% saturation (164 g/liter). The solution was applied at 0.3 ml/min to a phenyl-Sepharose column (1 by 0.8 cm) that had been pre-equilibrated with buffer A containing ammonium sulfate (30% saturation). The column was washed with the buffer, and then 40 ml of a linear gradient of ammonium sulfate, 30\u20130% saturation, in buffer A was applied. The fraction containing activity was concentrated and stored at 20 \u00b0C after adding glycerol to give 20% (v/v) concentration. N-terminal Amino Acid Sequencing One microgram of the purified enzyme was run on a 12.5% SDS\ufffepolyacrylamide Tricine gel (1 mm thick) (17), and then blotted onto a ProBlot\u00ae poly(vinylidene difluoride) membrane (Applied Biosystems). The protein band was examined with an Applied Biosystems model 477A protein sequencer, fitted with an on-line model 120A analyzer for the detection of phenylthiohydantoin-amino acids. Expression and Purification of the acpD Gene Product The acpD gene was obtained by PCR using genomic DNA of E. coli strain JM109 as the template. Pfu turbo DNA polymerase (Stratagene) and oligonucleotide primers (sense, 5-cggccatatgagcaaggtattagtt-3 containing an NdeI site; antisense, 5-gcgctcgagttatgcagaaacaat-3 con\ufffetaining a XhoI site) were used for PCR. The resulting DNA was sub\ufffecloned into the corresponding restriction site of pET22b to obtain a plasmid designated as pETacpD. The nucleotide sequence of the cloned DNA was confirmed by dideoxy sequencing. E. coli JM109 (DE3) har\ufffeboring pETacpD was grown at 37 \u00b0C in LB medium. Expression was induced with 0.5 mM isopropyl-1-thio--D-galactopyranoside (IPTG) for 3 h with an optical density at 600 nm of 0.6. The recombinant AcpD was purified by Q-Sepharose, Blue-Sepharose, Sephacryl S200, and GIGA\ufffePITE column chromatographies. Stoichiometry of Azo Reduction Using the standard assay system (2 ml) with 7.8 ng of the purified acpD gene product (designated as AcpD), the methyl red degradation rate and NADH consumption rate in the first 1.5 min were determined by monitoring the absorbance at 430 and 340 nm, respectively. Because the absorbance of methyl red at 340 nm was a linear function of the absorbance at 430 nm under the given conditions, the absorbance at 340 nm in the standard assay system was corrected by calculation. The molar absorption coefficients were as described above. Identification of Reaction Products Methyl red was incubated in 40 ml of the standard assay system for 10 min at 30 \u00b0C with the purified AcpD (20 \u0001g). For extraction of ABA, an aliquot (20 ml) of the reaction mixture was extracted with an equal volume of ethyl acetate three times, subsequent to adjusting the pH to 3 with 1 M HCl. The extracts were pooled and evaporated in a rotary evaporator. For DMPD, another aliquot (20 ml) was extracted with an equal volume of n-hexane three times, subsequent to adjusting the pH to 10 with 1 M NaOH. The extracts were pooled and evaporated in a rotary evaporator. Each residue was dissolved in 0.5 ml of acetonitrile. Each sample was diluted 200-fold with acetonitrile and then analyzed (20 \u0001l) with a Shimadzu HPLC system equipped with a model SPD-6AV variable wavelength detector (detection wavelength: 336 nm for ABA; 250 nm for DMPD) and a Jasco CrestPak ODS column (4.6 mm \u0004 150 mm). The mobile phase was composed of 25 mM phosphate and aceto\ufffenitrile, 5:95 (25 mM phosphate:acetonitrile). The flow rate was 0.8 ml/min. Overexpression and Purification of E. coli ACP The gene for ACP was amplified using genomic DNA of E. coli strain JM109 as the template for PCR. The forward primer included an EcoRI site before the start codon: 5-ctggaettcatgagcactatcga-3. The reverse primer included a HindIII restriction site after the stop codon: 5- ctgaagcttttacgcctggtggc-3. The PCR product was subcloned into the EcoRI/HindIII site of the pKK223\u20133 plasmid, and the resulting plasmid was designated as pKKacpP. E. coli JM109 was transformed with pKKacpP and grown at 37 \u00b0C with LB medium. After the cultures had been grown to an optical density at 600 nm of 0.6, expression was induced with 0.5 mM IPTG for 3 h. The purification procedure followed the method of Therisod et al. (18). In Vitro Assay for ACP Phosphodiesterase Activity The assay system comprised 50 mM Tris-HCl (pH 8.5), 0.02 mM MnCl2, 25 mM MgCl2,1mM dithiothreitol, 20 \u0001g of holo/apoACP, and 3.7 \u0001g of purified AcpD, in a final volume of 0.1 ml. After incubation at 35 \u00b0C for 12 h, a sample was analyzed by native PAGE (19). For the holoACP standard, 20 \u0001g of holo/apoACP was converted to holoACP under the conditions with 50 mM Tris-HCl (pH 8.8), 0.1 mM CoA, 25 mM MgCl2,1mM DTT, and 4.2 \u0001g of ACP synthase. ACP synthase was expressed and purified by a procedure based on that of Lambalot and Walsh (20). For the apoACP standard, Ser36-substituted ACP was used. PCR-based site-directed mutagenesis was carried out with substitution from Ser to Cys. The expression and purification procedure were the same as those for the wild type. In Vivo Assaying of ACP Phosphodiesterase Activity ACP was coexpressed with AcpD in E. coli and then analyzed by native PAGE. To obtain a plasmid (designated as pACYCacpP) that was compatible with pETacpD and produced ACP, the BamHI-HincII 0.6-kb DNA fragment of pACYC177 (21) was replaced by the 1.4-kb BamHI\ufffePvuI fragment (the cohesive end of the PvuI site was blunt-ended) from pKKacpP. The 1.4-kb fragment contained a tac promoter, the ACP gene, and rrnB ribosomal RNA transcription terminators. E. coli JM109 (DE3) was cotransformed with pACYCacpP and pETacpD or pET21a (for a control experiment). Expression was induced by the addition of IPTG as described above. Other Techniques Protein concentrations were determined using protein assay reagent (Bio-Rad), with bovine serum albumin as the standard. SDS-PAGE was carried out using 12.5% gels as described by Laemmli (22). The proteins on gels were stained using a Silver Stain Kit II (Wako Pure Chemicals, Osaka, Japan). The native molecular weight of the protein was deter\ufffemined by gel filtration on a Superdex 200 HR column (30 \u0004 1.0 cm) that had been equilibrated with Tris-buffered saline buffer (20 mM Tris-HCl (pH 7.5) and 0.15 M NaCl). Calibration of the column was carried out with the following proteins: alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa), all from Sigma."
  },
  "31449862": {
    "bleu": 97.59070152079886,
    "meteor": 97.12119248869755,
    "rouge1_p": 0.984118291347207,
    "rouge1_r": 0.9825041006014216,
    "rouge1_f": 0.9833105335157318,
    "rouge2_p": 0.976986301369863,
    "rouge2_r": 0.975382932166302,
    "rouge2_f": 0.9761839583903641,
    "rougeL_p": 0.9830230010952903,
    "rougeL_r": 0.9814106068890104,
    "rougeL_f": 0.9822161422708617,
    "cosine_similarity": 0.9648664932348902,
    "precision": 0.9594594594594594,
    "recall": 0.922077922077922,
    "ner_f1": 0.9403973509933775,
    "ner_tp": 142,
    "ner_fp": 6,
    "ner_fn": 12,
    "bertscore_p": 0.9699422121047974,
    "bertscore_r": 0.9473300576210022,
    "bertscore_f1": 0.9587410092353821,
    "bertscore_scibert_error": "The expanded size of the tensor (2718) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 2718].  Tensor sizes: [1, 512]",
    "len_gt": 10883,
    "len_jl": 10881,
    "GT": "2. Materials and methods 2.1. Materials Phanta super-fidelity DNA polymerase was purchased from Vazyme Biotech (Nanjing, China). Restriction enzymes (EcoRI, HindIII, XhoI) were from New England BioLabs (Ipswich, MA, USA). T4 DNA ligase was purchased from TAKARA (Japan). A genomic DNA extraction kit was purchased from QIAGEN (Germany). Other chemicals and reagents were purchased from local markets. 2.2. Screening and sequence analysis of azoreductase genes Through searching of the complete genome sequence of Streptomyces sp. S27 (unpublished), a gene (named azored2) coding a putative azoreductase was found. A phylogenetic tree of AzoRed2 and other azoreductases was constructed using MEGA 7.0 software. Multiple sequence alignments were carried out using the Clustal X program and exported using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/ ). 2.3. Heterologous expression and purification of azoreductase AzoRed2 The Streptomyces sp. S27 stored at \u221220 \u00b0C in our laboratory was inoculated into the liquid medium (KNO\u2083, 0.1%; K\u2082HPO\u2084, 0.05%; MgSO\u2084\u00b77H\u2082O, 0.05%; NaCl, 0.05%; FeSO\u2084\u00b77H\u2082O, 0.01%; Starch, 2.0%) and incubated at 37 \u00b0C with rotary shaking (180 rpm) for 24 h. The genomic DNA of S27 was extracted following the instructions of the genomic DNA extraction kit (QIAGEN, Germany). According to the nucleotide sequence of predicted azoreductase azored2, one pair of full-length primers was synthesized (azored2_F and azored2_R, Table S1). The recombinant plasmid was constructed using standard methods [32]. In this section, the expression vector and host strain were pET28a(+) and BL21(DE3), respectively. The constructed recombinant plasmid was sent to be sequenced (Sangon Biotech). E. coli BL21(DE3) containing the recombinant plasmid (pET28a-azored2) was cultivated at 37 \u00b0C in 100 mL LB liquid medium (+50 \u03bcg/mL Kanamycin) until the OD600 reached 0.4\u20130.7. Isopropyl-\u03b2-D-1-thiogalactopyranoside (IPTG) with a final concentration of 100 \u03bcM was added to the medium at 20 \u00b0C for 20 h to induce the AzoRed2 protein expression. The cells were harvested and washed with PBS buffer (pH 7.4) once, and then the cells were resuspended in 10 mL PBS buffer (pH 7.4). Induced cells were ruptured by sonication. After centrifugation (8000 \u00d7g) at 4 \u00b0C for 15 min, the supernatant of the cell lysis solution was loaded onto a Ni-NTA column (1 mL, GE Healthcare, USA). Unbound proteins were washed with PBS buffer (pH 7.4) containing 20 mM imidazole. The bound AzoRed2 protein was eluted using a linear imidazole gradient (20\u2013500 mM) in PBS buffer (pH 7.4). Finally, the purified AzoRed2 was analyzed by SDS-PAGE. 2.4. Enzyme characterization of AzoRed2 The azoreductase activity of AzoRed2 was determined using the method reported by Nakanishi et al. with minor modification [23]. Briefly, the assay system contained 25 \u03bcM methyl red, 250 \u03bcM NADH, and 5 \u03bcM FMN in phosphate buffer (100 mM) at 30 \u00b0C. The reaction was initiated by adding the AzoRed2 protein and was terminated by adding SDS with a final concentration of 1.0%. The decrease in the value of the absorbance at 430 nm was recorded. One unit of azoreductase activity was defined as the amount of enzyme required to degrade 1 \u03bcmol methyl red per minute. The molar adsorption coefficient of methyl red is 23.36 mM\u22121 cm\u22121. 2.4.1. Cofactor identification The non-covalently bound flavin in AzoRed2 was identified using UV\u2013vis adsorption spectra. Native purified AzoRed2 protein was used as a control and was further treated using 1.0% SDS (final concentration) to compare the changes in absorption peaks [33]. To determine the stoichiometric binding of AzoRed2 with FMN molecules, the purified AzoRed2 was incubated with excess amount of FMN at 4 \u00b0C for 30 min. After removing the unbound FMN by filtering through a Millipore Amicon Ultra 10,000 cutoff filter, the contents of protein and protein-bound FMN molecules in reconstituted AzoRed2-FMN complex were quantified. 2.4.2. Coenzyme preference determination Azoreductases use NADH or NADPH as a coenzyme to degrade azo dyes, and they may exhibit preference toward NADH and NADPH. Based on the initial reaction system, NADH was replaced by NADPH to determine the coenzyme preferences of AzoRed2. 2.4.3. Optimal temperature and thermostability To determine the optimal reaction temperature of AzoRed2, the reaction solution was incubated at temperatures ranging from 25 \u00b0C to 70 \u00b0C. The thermostability of AzoRed2 was determined by incubating AzoRed2 at different temperatures (25 \u00b0C\u201370 \u00b0C) for 1 h, and the residual activity was determined. 2.4.4. Optimal pH and pH stability The optimal reaction pH was studied over pH values ranging from 4.0 to 8.0 in different buffers (4.0\u20135.0, Na\u2082HPO\u2084-Citric acid buffer; 5.0\u20136.0, Sodium citrate buffer; and 6.0\u20138.0, Phosphate buffer). The procedure for examining pH stability was carried out by incubating the AzoRed2 protein in different pH buffers (pH 4.0 Na\u2082HPO\u2084-Citric acid buffer, pH 5.0 Sodium citrate buffer, pH 6.0 Sodium citrate buffer, pH 7.0 Phosphate buffer, pH 8.0 Tris-HCl buffer, pH 9.0 Tris-HCl buffer) at 25 \u00b0C for hours, and the residual activity was determined. 2.4.5. Substrate specificity Substrate specificity was determined using different azo dyes (Congo Red, Methyl Orange, Trypan Blue, Sudan Black B, and Direct Black 38), and methyl red was used as a control (activity defined as 100%). 2.4.6. Effect of detergents on AzoRed2 activity The effect of detergents on AzoRed2 activity was determined by adding 0.5% or 1.0% of various detergents (Tween 20, Tween 80, Triton X-100, SDS) to the reaction system. The group without adding any detergents was used as a control, and its catalytic activity was defined as 100%. 2.4.7. Effect of metal ions on AzoRed2 activity To estimate the influence of metal ions on AzoRed2 activity, the reaction solution was mixed with different metal ions (Ni\u00b2\u207a, Mg\u00b2\u207a, Ca\u00b2\u207a, Mn\u00b2\u207a, Cu\u00b2\u207a, Zn\u00b2\u207a, Co\u00b2\u207a, Fe\u00b2\u207a, and Fe\u00b3\u207a) and the chelating agent Na\u2082-EDTA. The activity of the solution without adding metal ions or Na\u2082-EDTA was defined as 100%. 2.4.8. Effect of organic solvents on AzoRed2 activity To estimate the effect of organic solvents on AzoRed2 activity, different organic solvents (DMSO, methanol, ethanol, acetonitrile, isopropanol, n-propanol, n-butanol, isoamyl alcohol, chloroform, n-hexane, and isooctane) were mixed with purified AzoRed2 solution, and the final organic solvent concentrations were 10% or 20%. The mixed solutions were incubated at 30 \u00b0C with rotary shaking (80 rpm) for 1 h, and the residual activities were finally determined. In order to reduce the effect of organic solvents on the determination of azoreductase activity, the hydrophobic organic solvents were removed by centrifugation from the mixture after incubation [34,35]. For hydrophilic organic solvents, the concentration of organic solvents was diluted to a low content (5%) before determining the azoreductase activity [34,35]. 2.4.9. Specific activity of AzoRed2 Under the optimal reaction conditions (25 \u03bcM methyl red, 250 \u03bcM NADH, and 5 \u03bcM FMN in Na\u2082HPO\u2084-Citric acid buffer (pH 5.0, 100 mM) at 55 \u00b0C), the activity of AzoRed2 was determined. The protein content was assayed according to the method of Bradford using bovine serum albumin as the standard protein. The relative activity was defined as the total azoreductase activity per mg protein. 2.5. Construction of a co-expression plasmid and co-expression of AzoRed2 and BsGDH The co-expression plasmid (pET28a-azored2-bsgdh) containing the azored2 gene and the B. subtilis gdh gene (WP_003246720.1) was constructed as described in Scheme 1 [27]. A pair of oligonucleotide primers (sd-as-bsgdh_F and sd-as-bsgdh_R, Table S1) was designed and synthesized. The PCR product of sd-as-bsgdh was double digested and then ligated to the plasmid pET28a-azored2. The recombinant BL21(DE3) strain containing the co-expression pET28a-azored2-bsgdh plasmid was used to express AzoRed2 and BsGDH as described above. SDS-PAGE was used to determine the expression levels of AzoRed2 and BsGDH. The azoreductase activity (NADH as the coenzyme) of the cell lysate supernatant was determined as described above. The GDH activity (NAD\u207a as the coenzyme) of the cell lysate supernatant was assayed as in the report described [27]. The coupling enzymatic activity of the supernatant toward methyl red (NAD\u207a as the coenzyme) was also determined. After determining the protein content, the relative activities were calculated. 2.6. Methyl red degradation using the whole-cell biocatalyst The wet co-expression cells containing AzoRed2 and BsGDH were used to study the efficiency of degrading methyl red. The initial reaction system contained 2.5 mg/mL biocatalyst, 100 \u03bcM methyl red, 20 mM glucose, and pH 6.0 sodium citrate buffer (100 mM). The mixed reaction system was incubated under 30 \u00b0C with rotary shaking (80 rpm). The reaction solution was sampled, and the residual methyl red was quantified at various intervals, after which, the decolorization was determined. The decolorization of the solution represented the degradation rate of methyl red, and the decolorization determining formula was as follows: \ud835\udc37 \ud835\udc52 \ud835\udc50 \ud835\udc5c \ud835\udc59 \ud835\udc5c \ud835\udc5f \ud835\udc56 \ud835\udc67 \ud835\udc4e \ud835\udc61 \ud835\udc56 \ud835\udc5c \ud835\udc5b = 1 \u2212 Residual methyl red Initial methyl red Decolorization=1\u2212 Initial methyl red Residual methyl red \u200b 2.6.1. Optimization of reaction conditions 2.6.1.1. Effect of additional concentration of NAD\u207a. Based on the reaction conditions, the effect of additional NAD\u207a amounts was studied. The additional amounts were set as 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM. 2.6.1.2. Effect of glucose concentration. To study whether glucose concentration had a significant effect on the degradation efficiency of methyl red, the glucose concentration was varied (5, 10, 20, 30, 40, and 60 mM), while other factors remained unchanged at the initial conditions. 2.6.1.3. Effect of biocatalyst loading. Under the initial reaction conditions, biocatalyst loading was further used as the variable to carry out the degradation reaction. The loading amounts of wet whole-cell biocatalysts were 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 mg/mL. 2.6.1.4. Effect of methyl red concentration. After evaluating the effects of additional amounts of NAD\u207a, biocatalyst loadings, and glucose concentrations, the substrate tolerance of the whole-cell biocatalyst was tested under optimal conditions (no NAD\u207a addition, 5.0 mg/mL wet whole-cell biocatalyst and 10 mM glucose). The concentrations of methyl red in the medium were increased to 125, 150, 175, 200, 225, and 250 \u03bcM. 2.6.2. Batch biodegradation of methyl red using the whole-cell biocatalyst In this section, the wet whole-cell biocatalyst was used to determine its reusability, and the reaction system was as follows: 5.0 mg/mL biocatalyst, 250 \u03bcM methyl red and 10 mM glucose in pH 6.0 sodium citrate buffer (100 mM). The dye removal process was assessed during 2.0 h per batchwise cycle.",
    "LLM": "2. Materials and methods 2.1. Materials Phanta super-fidelity DNA polymerase was purchased from Vazyme Biotech (Nanjing, China). Restriction enzymes (EcoRI, HindIII, XhoI) were from New England BioLabs (Ipswich, MA, USA). T4 DNA ligase was purchased from TAKARA (Japan). A genomic DNA extraction kit was purchased from QIAGEN (Germany). Other chemicals and reagents were purchased from local markets. 2.2. Screening and sequence analysis of azoreductase genes Through searching of the complete genome sequence of Streptomyces sp. S27 (unpublished), a gene (named azored2) coding a putative azoreductase was found. A phylogenetic tree of AzoRed2 and other azoreductases was constructed using MEGA 7.0 software. Multiple sequence alignments were carried out using the Clustal X program and exported using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/). 2.3. Heterologous expression and purification of azoreductase AzoRed2 The Streptomyces sp. S27 stored at \u221220 \u00b0C in our laboratory was inoculated into the liquid medium (KNO3, 0.1%; K2HPO4, 0.05%; MgSO4\u00b77H2O, 0.05%; NaCl, 0.05%; FeSO4\u00b77H2O, 0.01%; Starch, 2.0%) and incubated at 37 \u00b0C with rotary shaking (180 rpm) for 24 h. The genomic DNA of S27 was extracted following the instructions of the genomic DNA extraction kit (QIAGEN, Germany). According to the nucleotide sequence of predicted azoreductase azored2, one pair of full-length primers was synthesized (azored2_F and azored2_R, Table S1). The recombinant plasmid was constructed using standard methods [32]. In this section, the expression vector and host strain were pET28a(+) and BL21(DE3), respectively. The constructed recombinant plasmid was sent to be sequenced (Sangon Biotech). E. coli BL21(DE3) containing the recombinant plasmid (pET28a-azored2) was cultivated at 37 \u00b0C in 100 mL LB liquid medium (+50 \u03bcg/mL Kanamycin) until the OD600 reached 0.4\u20130.7. Isopropyl-\u03b2-D-1-thiogalactopyranoside (IPTG) with a final concentration of 100 \u03bcM was added to the medium at 20 \u00b0C for 20 h to induce the AzoRed2 protein expression. The cells were harvested and washed with PBS buffer (pH 7.4) once, and then the cells were resuspended in 10 mL PBS buffer (pH 7.4). Induced cells were ruptured by sonication. After centrifugation (8000 \u00d7g) at 4 \u00b0C for 15 min, the supernatant of the cell lysis solution was loaded onto a Ni-NTA column (1 mL, GE Healthcare, USA). Unbound proteins were washed with PBS buffer (pH 7.4) containing 20 mM imidazole. The bound AzoRed2 protein was eluted using a linear imidazole gradient (20\u2013500 mM) in PBS buffer (pH 7.4). Finally, the purified AzoRed2 was analyzed by SDS-PAGE. 2.4. Enzyme characterization of AzoRed2 The azoreductase activity of AzoRed2 was determined using the method reported by Nakanishi et al. with minor modification [23]. Briefly, the assay system contained 25 \u03bcM methyl red, 250 \u03bcM NADH, and 5 \u03bcM FMN in phosphate buffer (100 mM) at 30 \u00b0C. The reaction was initiated by adding the AzoRed2 protein and was terminated by adding SDS with a final concentration of 1.0%. The decrease in the value of the absorbance at 430 nm was recorded. One unit of azoreductase activity was defined as the amount of enzyme required to degrade 1 \u03bcmol methyl red per minute. The molar adsorption coefficient of methyl red is 23.36 mM\u22121 cm\u22121. 2.4.1. Cofactor identification The non-covalently bound flavin in AzoRed2 was identified using UV\u2013vis adsorption spectra. Native purified AzoRed2 protein was used as a control and was further treated using 1.0% SDS (final concentration) to compare the changes in absorption peaks [33]. To determine the stoichiometric binding of AzoRed2 with FMN molecules, the purified AzoRed2 was incubated with excess amount of FMN at 4 \u00b0C for 30 min. After removing the unbound FMN by filtering through a Millipore Amicon Ultra 10,000 cutoff filter, the contents of protein and protein-bound FMN molecules in reconstituted AzoRed2-FMN complex were quantified. 2.4.2. Coenzyme preference determination Azoreductases use NADH or NADPH as a coenzyme to degrade azo dyes, and they may exhibit preference toward NADH and NADPH. Based on the initial reaction system, NADH was replaced by NADPH to determine the coenzyme preferences of AzoRed2. 2.4.3. Optimal temperature and thermostability To determine the optimal reaction temperature of AzoRed2, the reaction solution was incubated at temperatures ranging from 25 \u00b0C to 70 \u00b0C. The thermostability of AzoRed2 was determined by incubating AzoRed2 at different temperatures (25 \u00b0C\u201370 \u00b0C) for 1 h, and the residual activity was determined. 2.4.4. Optimal pH and pH stability The optimal reaction pH was studied over pH values ranging from 4.0 to 8.0 in different buffers (4.0\u20135.0, Na2HPO4-Citric acid buffer; 5.0\u20136.0, Sodium citrate buffer; and 6.0\u20138.0, Phosphate buffer). The procedure for examining pH stability was carried out by incubating the AzoRed2 protein in different pH buffers (pH 4.0 Na2HPO4-Citric acid buffer, pH 5.0 Sodium citrate buffer, pH 6.0 Sodium citrate buffer, pH 7.0 Phosphate buffer, pH 8.0 Tris-HCl buffer, pH 9.0 Tris-HCl buffer) at 25 \u00b0C for hours, and the residual activity was determined. 2.4.5. Substrate specificity Substrate specificity was determined using different azo dyes (Congo Red, Methyl Orange, Trypan Blue, Sudan Black B, and Direct Black 38), and methyl red was used as a control (activity defined as 100%). 2.4.6. Effect of detergents on AzoRed2 activity The effect of detergents on AzoRed2 activity was determined by adding 0.5% or 1.0% of various detergents (Tween 20, Tween 80, Triton X-100, SDS) to the reaction system. The group without adding any detergents was used as a control, and its catalytic activity was defined as 100%. 2.4.7. Effect of metal ions on AzoRed2 activity To estimate the influence of metal ions on AzoRed2 activity, the reaction solution was mixed with different metal ions (Ni2+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, Co2+, Fe2+, and Fe3+) and the chelating agent Na2-EDTA. The activity of the solution without adding metal ions or Na2-EDTA was defined as 100%. 2.4.8. Effect of organic solvents on AzoRed2 activity To estimate the effect of organic solvents on AzoRed2 activity, different organic solvents (DMSO, methanol, ethanol, acetonitrile, isopropanol, n-propanol, n-butanol, isoamyl alcohol, chloroform, n-hexane, and isooctane) were mixed with purified AzoRed2 solution, and the final organic solvent concentrations were 10% or 20%. The mixed solutions were incubated at 30 \u00b0C with rotary shaking (80 rpm) for 1 h, and the residual activities were finally determined. In order to reduce the effect of organic solvents on the determination of azoreductase activity, the hydrophobic organic solvents were removed by centrifugation from the mixture after incubation [34,35]. For hydrophilic organic solvents, the concentration of organic solvents was diluted to a low content (5%) before determining the azoreductase activity [34,35]. 2.4.9. Specific activity of AzoRed2 Under the optimal reaction conditions (25 \u03bcM methyl red, 250 \u03bcM NADH, and 5 \u03bcM FMN in Na2HPO4-Citric acid buffer (pH 5.0, 100 mM) at 55 \u00b0C), the activity of AzoRed2 was determined. The protein content was assayed according to the method of Bradford using bovine serum albumin as the standard protein. The relative activity was defined as the total azoreductase activity per mg protein. 2.5. Construction of a co-expression plasmid and co-expression of AzoRed2 and BsGDH The co-expression plasmid (pET28a-azored2-bsgdh) containing the azored2 gene and the B. subtilis gdh gene (WP_003246720.1) was constructed as described in Scheme 1 [27]. A pair of oligonucleotide primers (sd-as-bsgdh_F and sd-as-bsgdh_R, Table S1) was designed and synthesized. The PCR product of sd-as-bsgdh was double digested and then ligated to the plasmid pET28a-azored2. The recombinant BL21(DE3) strain containing the co-expression pET28a-azored2-bsgdh plasmid was used to express AzoRed2 and BsGDH as described above. SDS-PAGE was used to determine the expression levels of AzoRed2 and BsGDH. Scheme 1. Construction of recombinant plasmid coupled AzoRed2 and BsGDH. The azoreductase activity (NADH as the coenzyme) of the cell lysate supernatant was determined as described above. The GDH activity (NAD+ as the coenzyme) of the cell lysate supernatant was assayed as in the report described [27]. The coupling enzymatic activity of the supernatant toward methyl red (NAD+ as the coenzyme) was also determined. After determining the protein content, the relative activities were calculated. 2.6. Methyl red degradation using the whole-cell biocatalyst The wet co-expression cells containing AzoRed2 and BsGDH were used to study the efficiency of degrading methyl red. The initial reaction system contained 2.5 mg/mL biocatalyst, 100 \u03bcM methyl red, 20 mM glucose, and pH 6.0 sodium citrate buffer (100 mM). The mixed reaction system was incubated under 30 \u00b0C with rotary shaking (80 rpm). The reaction solution was sampled, and the residual methyl red was quantified at various intervals, after which, the decolorization was determined. The decolorization of the solution represented the degradation rate of methyl red, and the decolorization determining formula was as follows: Decolorization \u00bc 1\u2212Residual methyl red Initial methyl red 2.6.1. Optimization of reaction conditions 2.6.1.1. Effect of additional concentration of NAD+. Based on the reaction conditions, the effect of additional NAD+ amounts was studied. The additional amounts were set as 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM. 2.6.1.2. Effect of glucose concentration. To study whether glucose concentration had a significant effect on the degradation efficiency of methyl red, the glucose concentration was varied (5, 10, 20, 30, 40, and 60 mM), while other factors remained unchanged at the initial conditions. 2.6.1.3. Effect of biocatalyst loading. Under the initial reaction conditions, biocatalyst loading was further used as the variable to carry out the degradation reaction. The loading amounts of wet whole-cell biocatalysts were 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 mg/mL. 2.6.1.4. Effect of methyl red concentration. After evaluating the effects of additional amounts of NAD+, biocatalyst loadings, and glucose concentrations, the substrate tolerance of the whole-cell biocatalyst was tested under optimal conditions (no NAD+ addition, 5.0 mg/mL wet whole-cell biocatalyst and 10 mM glucose). The concentrations of methyl red in the medium were increased to 125, 150, 175, 200, 225, and 250 \u03bcM. 2.6.2. Batch biodegradation of methyl red using the whole-cell biocatalyst In this section, the wet whole-cell biocatalyst was used to determine its reusability, and the reaction system was as follows: 5.0 mg/mL biocatalyst, 250 \u03bcM methyl red and 10 mM glucose in pH 6.0 sodium citrate buffer (100 mM). The dye removal process was assessed during 2.0 h per batchwise cycle."
  },
  "30465830": {
    "bleu": 84.5737624175461,
    "meteor": 78.51059091924604,
    "rouge1_p": 0.8832236842105263,
    "rouge1_r": 0.9086294416243654,
    "rouge1_f": 0.8957464553794829,
    "rouge2_p": 0.7884773662551441,
    "rouge2_r": 0.8111769686706182,
    "rouge2_f": 0.7996661101836394,
    "rougeL_p": 0.881578947368421,
    "rougeL_r": 0.9069373942470389,
    "rougeL_f": 0.8940783986655546,
    "cosine_similarity": 0.7712564413622076,
    "precision": 0.7676767676767676,
    "recall": 0.6972477064220184,
    "ner_f1": 0.7307692307692308,
    "ner_tp": 76,
    "ner_fp": 23,
    "ner_fn": 33,
    "bertscore_p": 0.8658721446990967,
    "bertscore_r": 0.9088046550750732,
    "bertscore_f1": 0.8875535130500793,
    "bertscore_scibert_error": "The expanded size of the tensor (1963) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1963].  Tensor sizes: [1, 512]",
    "len_gt": 7032,
    "len_jl": 7176,
    "GT": "2. Materials and methods 2.1. Bacterial strains, plasmids and culture medium Acid Brilliant Scarlet GR (ABS) was purchased from the Adamas Reagent, Co. Ltd., Shanghai, China. Molecular biology enzymes and kits, such as restriction endonucleases, a Genomic DNA isolation Kit, an RNA isolation Kit, DNase I, the PrimeScript RT reagent Kit with gDNA Eraser, and SYBR\u00ae Premix DimerEraser\u2122 were purchased from Takara (Takara, Dalian, China). The pET-30a plasmid was purchased from Novagen (USA). E. coli DH5\u03b1 cells and E. coli BL21 (DE3) were purchased from Tiangen (China). All chemicals used were of analytical grade. Sea salt-defined media was used to isolate halophilic bacteria [14]. 2.2. Isolation and sequencing of halophilic bacteria The Halomonas sp. strain GT was isolated through the standard dilution plating technique. The isolates were initially screened for their ability to decolorize ABS in the sea salt-defined media agar plates amended with 50 mg L\u207b\u00b9 dye. The isolates showing clear zones on agar plates were further selected for decolorization studies. The genome was sequenced on the PacBio RSII sequencing platforms. Genomic DNA from Halomonas sp. strain GT was used to construct a PacBio SMRTbell\u2122 library, which generated 80,580 filtered sub-reads totaling 859.7 Mbp. The average length is 10,669 bp. The complete genome was sequenced using Pacific Biosciences (PacBio) technology (Major Bio, China). The PacBio reads were assembled using HGAP (version: 2.1.1) [15]. Glimmer version 3.0.2 (http://ccb.jhu.edu/software/glimmer/index.shtml ) was used to predict coding sequences (CDS). Gene identification and annotation were conducted via BLASTp [16] search against the NCBI-NR and Kyoto Encyclopedia of Genes and Genomes (KEGG) database [17]. The tRNAScanSE tool and barrnap 0.7 (http://www.vicbioinformatics.com/software.barrnap.shtml ) were used to identify the tRNA and rRNA genes. The whole genome sequence of Halomonas sp. strain GT has been deposited in GenBank under accession number CP014226. 2.3. Cloning and construction of recombinant azo in E. coli The azoreductase gene of the Halomonas sp. strain GT (named azoG) was cloned by PCR using the following primer pair: 5\u2032 TCCATGGATGACCACTCGTGCAC 3\u2032 and 5\u2032 GTCTGGAATTC ACTAGAGTGTTTCG 3\u2032 (the underlining indicates the added NcoI and EcoRI site). The PCR product was purified, cloned into the PMD-19T vector and transformed into E. coli DH5\u03b1. The plasmids were extracted and cleaved with NcoI and EcoRI, then inserted into pET30a (+). The plasmid was transformed into E. coli BL21 (DE3). Protein expression was induced for 6 h with 0.5 mM IPTG when the culture turbidity reached 0.6 (OD600). 2.4. Phylogenetic analysis The BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/ ) was used for azoG amino acid sequence homology searches. The neighbor-joining methods (MEGA 5.0) were used to construct the phylogenetic tree. 2.5. Decolorization analyses Halomonas sp. strain GT was collected from the sea salt-defined media at 30 \u00b0C by centrifugation (4000g at 4 \u00b0C, 5 min), and rinsed twice using sea salt-defined media. The effects of different NaCl concentration (1%, 5%, 10%, 15%, 20%), on the decolorization rate were tested. The initial dye concentration was 100 mg L\u207b\u00b9. The decolorization tests were carried out in sea salt-defined media at 10% salinity with yeast extract as electron donor (1 g L\u207b\u00b9), at 30 \u00b0C, pH 7.5. Bacterial samples were withdrawn from the culture to detect the decolorization ratio, azoG gene expression and azoG activity. The culture media were centrifuged for removing the bacterial cell and the supernatant was used to measure the absorbance. The absorbance was measured at 2, 4, 6, 10, 14 h. The decolorization ratio was calculated according to equation: Decolorization (%) = (A0 \u2212 At)/A0 \u00d7 100, where A0 and At represent the initial absorbance of the dye at 510 nm and the absorbance after incubation. 2.6. Quantitative real-time PCR (Q-PCR) analysis of azoG gene expression Q-PCR was used to detect azoG gene expression. Total RNA was extracted using RNAiso plus isolation Kit (Takara, Dalian, China) according to the manufacturer's instructions. Reverse transcription of RNA samples was generated. The cDNA samples were used for Q-PCR, which was conducted in an Eppendorf Mastercycler Real-time thermal cycler using SYBR Premix Ex-Taq II according to the manufacturer's directions. The primers for azoG were 5\u2032 GTGATGTAGTGGCTAATGCGTTAC 3\u2032 and 5\u2032 GGCGTCTGGCTGTCTAATTCAG 3\u2032. 16S gene was used as reference gene. The 16S primer were 5\u2032 CTGGGACGGAATGCCTAAAT 3\u2032 and 5\u2032 GTAGTCCACGCCGTAAACGATG 3\u2032. The PCR reaction condition were as follows: 35 cycles of 5 s at 96 \u00b0C, 20 s at 60 \u00b0C and 30 s at 72 \u00b0C. Gene expression levels were calculated using the 2-\u0394\u0394CT method. 2.7. Overexpression of azoG and SDS-PAGE analysis Cells were collected, suspended in phosphate buffer (pH 7.0) and disrupted by sonication. Thereafter, the cell debris was separated by centrifugation (10,000 \u00d7g, 30 min, 4 \u00b0C), and the azoG was purified by Ni\u00b2\u207a-affinity chromatography. SDS-PAGE (12.5%, w/v) was used to verify the purity of the protein. Bovine serum albumin was used as the standard protein to confirm the molecular mass of the protein. 2.8. Biochemical characterization of enzymes The azoreductase activity of azoG was tested using a UV\u2013vis spectrophotometer [12]. Standard reactions starting with appropriate purified azoG were carried out in 1 ml final volume containing 50 mM pH 7.0 sodium phosphate buffer, 150 \u03bcM NADH, 50 \u03bcM FMN and 25 \u03bcM methyl red at room temperature. The reaction was initiated by the addition of NADH prepared in 50 mM sodium phosphate buffer (pH 7.0) to a final concentration of 1 mM. Azoreductase activity was measured by the decrease in absorbance at 430 nm over a 1 min period using UV/VIS spectrophotometer. The extinction coefficient for methyl red at 430 nm was 23,360 M\u207b\u00b9 cm\u207b\u00b9. The enzyme activity was defined as the decrease in azo dye concentration (\u03bcM) per min. The Bradford method was used to detect protein concentrations [18]. The effect of salt on azoG activity was examined in 50 mM phosphate buffer (pH 7.0) containing 0\u201320% NaCl (w/v). The halo-stability of azoG was tested at 0%, 5%, 10% and 20% salinity. The residual activities of azoG were detected using the above method after incubation in buffers with 0\u201320% NaCl (w/v) at 4 \u00b0C for 1 h. The effect of temperature on AzoG was tested by assaying enzyme activity in a temperature range of 10 \u00b0C, 20 \u00b0C, 30 \u00b0C, 40 \u00b0C, 50 \u00b0C. The thermostability of the enzyme was also determined after the incubation of azoG at different temperature for 1 h. Residual reductase activity was determined using the above method. The effect of pH on azoG was analyzed in 50 mM buffer under different pH conditions. In order to find the optimal pH, azoreductase activity was measured in 50 mM buffer of glycine, citrate and phosphate, within the pH range of 4\u201310. pH stability was tested with above standard method after incubation at corresponding pH values at 4 \u00b0C for 1 h.",
    "LLM": "{\"methods_text\":\"2. Materials and methods\\n2.1. Bacterial strains, plasmids and culture medium\\nAcid Brilliant Scarlet GR (ABS) was purchased from the Adamas Re\ufffeagent, Co. Ltd., Shanghai, China. Molecular biology enzymes and kits,\\nsuch as restriction endonucleases, a Genomic DNA isolation Kit, an RNA\\nisolation Kit, DNase I, the PrimeScript RT reagent Kit with gDNA Eraser,\\nand SYBR \u00aePremix DimerEraser\u2122 were purchased from Takara (Takara,\\nDalian, China). The pET-30a plasmid was purchased from Novagen\\n(USA). E. coli DH5\u03b1 cells and E. coli BL21 (DE3) were purchased from\\nTiangen (China). All chemicals used were of analytical grade. Sea salt\ufffedefined media was used to isolate halophilic bacteria [14].\\n2.2. Isolation and sequencing of halophilic bacteria\\nThe Halomonas sp. strain GT was isolated through the standard dilu\ufffetion plating technique. The isolates were initially screened for their abil\ufffeity to decolorize ABS in the sea salt-defined media agar plates amended\\nwith 50 mg L\u22121 dye. The isolates showing clear zones on agar plates\\nwere further selected for decolorization studies. The genome was se\ufffequenced on the PacBio RSII sequencing platforms. Genomic DNA from\\nHalomonas sp. strain GT was used to construct a PacBio SMRTbellTM li\ufffebrary, which generated 80,580 filtered sub-reads totaling 859.7 Mbp.\\nThe average length is 10,669 bp. The complete genome was sequenced\\nusing Pacific Biosciences (PacBio) technology (Major Bio, China). The\\nPacBio reads were assembled using HGAP (version: 2.1.1) [15]. Glimmer\\nversion 3.0.2 (http://ccb.jhu.edu/software/glimmer/index.shtml) was\\nused to predict coding sequences (CDS). Gene identification and anno\ufffetation were conducted via BLASTp [16] search against the NCBI-NR\\nand Kyoto Encyclopedia of Genes and Genomes (KEGG) database [17].\\nThe tRNAScanSE tool and barrnap 0.7 (http://www.vicbioinformatics.\\ncom/software.barrnap.shtml) were used to identify the tRNA and\\nrRNA genes. The whole genome sequence of Halomonas sp. strain GT\\nhas been deposited in GenBank under accession number CP014226.\\n2.3. Cloning and construction of recombinant azo in E. coli\\nThe azoreductase gene of the Halomonas sp. strain GT (named azoG)\\nwas cloned by PCR using the following primer pair: 5\u2032 TCCATGGATGACC\\nACTCGTGCAC 3\u2032 and 5\u2032GTCTGGAATTC ACTAGAGTGTTTCG 3\u2032 (the\\nunderlining indicates the added Nco I and EcoR I site). The PCR product\\nwas purified, cloned into the PMD-19T vector and transformed into\\nE. coli DH5\u03b1. The plasmids were extracted and cleaved with Nco I and\\nEcoR I, then inserted into pET30a (+). The plasmid was transformed\\ninto E. coli BL21 (DE3). Protein expression was induced for 6 h with\\n0.5 mM IPTG when the culture turbidity reached 0.6 (OD600).\\n2.4. Phylogenetic analysis\\nThe BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) was used\\nfor azoG amino acid sequence homology searches. The neighbor-joining\\nmethods (MEGA 5.0) were used to construct the phylogenetic tree.\\n2.5. Decolorization analyses\\nHalomonas sp. strain GT was collected from the sea salt-defined\\nmedia at 30 \u00b0C by centrifugation (4000g at 4 \u00b0C, 5 min), and rinsed\\ntwice using sea salt-defined media. The effects of different NaCl\\nconcentration (1%, 5%, 10%, 15%, 20%), on the decolorization rate were\\ntested. The initial dye concentration was 100 mg L\u22121\\n. The decolorization\\ntests were carried out in sea salt-defined media at 10% salinity with\\nyeast extract as electron donor (1 g L\u22121\\n), at 30 \u00b0C, pH 7.5. Bacterial sam\ufffeples were withdrawn from the culture to detect the decolorization ratio,\\nazoG gene expression and azoG activity. The culture media were centri\ufffefuged for removing the bacterial cell and the supernatant was used to\\nmeasure the absorbance. The absorbance was measured at 2, 4, 6, 10,\\n14 h. The decolorization ratio was calculated according to equation:\\nDecolorization (%) = (A0 \u2212 At)/A0 \u00d7 100, where A0 and At represent\\nthe initial absorbance of the dye at 510 nm and the absorbance after in\ufffecubation absorbance.\\n2.6. Quantitative real-time PCR (Q-PCR) analysis of azoG gene expression\\nQ-PCR was used to detect azoG gene expression. Total RNA was ex\ufffetracted using RNAiso plus isolation Kit (Takara, Dalian, China) according\\nto the manufacturer's instructions. Reverse transcription of RNA sam\ufffeples was generated. The cDNA samples were used for Q-PCR, which\\nwas conducted in an Eppendorf Mastercycler Real-time thermal cycler\\nusing SYBR Premix Ex-Taq II according to the manufacturer's directions.\\nThe primers for azoG were 5\u2032 GTGATGTAGTGGCTAATGCGTTAC 3\u2032 and 5\u2032\\nGGCGTCTGGCTGTCTAATTCAG 3\u2032. 16S gene was used as reference gene.\\nThe 16S primer were 5\u2032 CTGGGACGGAATGCCTAAAT 3\u2032 and 5\u2019 GTAGTC\\nCACGCCGTAAACGATG 3\u2032. The PCR reaction condition were as follows:\\n35 cycles of 5 s at 96 \u00b0C, 20 s at 60 \u00b0C and 30 s at 72 \u00b0C. Gene expression\\nlevels were calculated using the 2-\u0394\u0394CTmethod.\\n2.7. Overexpression of azoG and SDS-PAGE analysis\\nCells were collected, suspended in phosphate buffer (pH 7.0) and\\ndisrupted by sonication. Thereafter, the cell debris was separated by\\ncentrifugation (10,000 \u00d7g, 30 min, 4 \u00b0C), and the azoG was purified by\\nNi2+-affinity chromatography. SDS-PAGE (12.5%, w/v) was used to ver\ufffeify the purity of the protein. Bovine serum albumin was used as the\\nstandard protein to confirm the molecular mass of the protein.\\n2.8. Biochemical characterization of enzymes\\nThe azoredutase activity of azoG was tested using a UV\u2013vis spectro\ufffephotometer [12]. Standard reactions starting with appropriate purified\\naZoG were carried out in 1 ml final volume containing 50 mM pH 7.0 so\ufffedium phosphate buffer, 150 \u03bcM NADH, 50 \u03bcM FMN and 25 \u03bcM methyl red\\nat room temperature. The reaction was initiated by the addition of NADH\\nprepared in 50 mM sodium phosphate buffer (pH 7.0) to a final concen\ufffetration of 1 mM. Azoreductase activity was measured by the decrease in\\nabsorbance at 430 nm over a 1 min period using UV/VIS spectrophotom\ufffeeter. The extinction coefficient for methyl red at 430 nm was\\n23,360 M\u22121 cm\u22121\\n. The enzyme activity was defined as the decrease in\\naZo dye concentration (\u03bcM) per min. The Bradford method was used to\\ndetect protein concentrations [18]. The effect of salt on azoG activity\\nwas examined in 50 mM phosphate buffer (pH 7.0) containing 0\u201320%\\nNaCl (w/v). The halo-stability of azoG was tested at 0%, 5%, 10% and 20%\\nsalinity. The residual activities of azoG were detected using the above\\nmethod after incubation in buffers with 0\u201320% NaCl (w/v) at 4 \u00b0C for 1 h.\\nThe effect of temperature on AzoG was tested by assaying enzyme\\nactivity in a temperature range of 10 \u00b0C, 20 \u00b0C, 30 \u00b0C, 40 \u00b0C, 50 \u00b0C. The\\nthermostability of the enzyme was also determined after the incubation\\nof azoG at different temperature for 1 h. Residual reductase activity was\\ndetermined using the above method.\\nThe effect of pH on azoG was analyzed in 50 mM buffer under differ\ufffeent pH conditions. In order to find the optimal pH, azoreductase activity\\nwas measured in 50 mM buffer of glycine, citrate and phosphate, within\\nthe pH range of 4\u201310. pH stability was tested with above standard\\nmethod after incubation at corresponding pH values at 4 \u00b0C for 1 h.\"}"
  },
  "20057057": {
    "bleu": 98.71299871299871,
    "meteor": 98.71262953821017,
    "rouge1_p": 0.9845679012345679,
    "rouge1_r": 0.9845679012345679,
    "rouge1_f": 0.9845679012345679,
    "rouge2_p": 0.9675425038639877,
    "rouge2_r": 0.9675425038639877,
    "rouge2_f": 0.9675425038639877,
    "rougeL_p": 0.9830246913580247,
    "rougeL_r": 0.9830246913580247,
    "rougeL_f": 0.9830246913580247,
    "cosine_similarity": 0.964430979014248,
    "precision": 0.9367088607594937,
    "recall": 0.9736842105263158,
    "ner_f1": 0.9548387096774194,
    "ner_tp": 74,
    "ner_fp": 5,
    "ner_fn": 2,
    "bertscore_p": 0.9902132749557495,
    "bertscore_r": 0.9786495566368103,
    "bertscore_f1": 0.9844784736633301,
    "bertscore_scibert_error": "The expanded size of the tensor (1005) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1005].  Tensor sizes: [1, 512]",
    "len_gt": 3946,
    "len_jl": 3943,
    "GT": "2. Materials and methods 2.1. Site-directed mutagenesis and transformation The pET28b(+) plasmid vector containing the sequence of paazor1 was isolated from a 6 ml culture of Escherichia coli BL21 (DE3) using a QIAprep Spin Miniprep Kit (Qiagen). Site-directed mutagenesis was achieved using a QuikChange II kit (Stratagene). The reaction mixture (50 \u00b5l), which contained 5 \u00b5l 10\u00d7 reaction buffer, 50 ng pET28b(+) plasmid as template, 125 ng of both Y131F forward (5\u2032-CGTCGCCCAGTTCCGGCCGCTGC-3\u2032) and Y131F reverse (5\u2032-GCAGCGGCCGGAACTGGGCGACG-3\u2032) primers and 1 \u00b5l dNTP mix, was subjected to thermo-cycling: 30 s at 368 K and 12 cycles of 30 s at 368 K, 1 min at 328 K and 6 min at 341 K, followed by 2 min on ice. The reaction mixture was digested (10 U DpnI, 1 h, 310 K) to cleave all template DNA before being purified by gel electrophoresis in ethidium-bromide-stained agarose [1.0%(w/v)]. The sequence of the insert was confirmed and the pET28b(+)-Y131F plasmid vector was transformed into E. coli BL21 (DE3) pLysS as described previously (Wang et al., 2007). 2.2. Protein production, purification and characterization Like wild-type paAzoR1, the Y131F mutant was expressed with a hexahistidine tag; the protein was then purified via affinity purification as described previously (Wang et al., 2007). Spectra were recorded from 600 to 300 nm and the enzymic activities were determined as described previously (Wang et al., 2007). Thermostability was determined by incubating the enzyme (10 min) from 277 to 363 K followed by 1 min on ice before adding methyl red and NADPH and measuring the loss of absorbance as described in Wang et al. (2007). Initial velocities were also measured at different concentrations of substrate and cofactor [from 5 to 40 mM methyl red and from 0.1 to 2.0 mM NAD(P)H], while the concentration of the other substrate was kept constant (Wang et al., 2007). In each case, linearity was established. Apparent Km and Vmax values were obtained from Lineweaver\u2013Burk plots using the initial linear rates expressed in \u00b5M s\u207b\u00b9. 2.3. Protein crystallization and structural determination Crystals of the mutant enzyme in the presence of methyl red (Y131F_MRE) were grown by sitting-drop vapour diffusion. Protein drops were prepared by mixing equal volumes (1 \u00b5l) of protein solution (23 mg ml\u207b\u00b9 in sterile water with 2 mM methyl red) with mother liquor (Molecular Dimensions JCSG-plus screen condition E8; 0.1 M sodium acetate pH 4.5, 1.0 M diammonium hydrogen phosphate) in a 24-well CrystalPlate (Axygen Bioscience). Crystals were briefly transferred to a cryoprotectant solution consisting of 1:3 glycerol:mother liquor prior to freezing in liquid nitrogen. Data were collected on beamline ID14-EH4 at the European Synchrotron Radiation Facility (ESRF, Grenoble) using a Quantum 315 ADSC CCD detector. Data were integrated with iMOSFLM v.0.5.2 (Leslie, 1992) and scaled and merged with SCALA (Collaborative Computational Project, Number 4, 1994). The structure was solved using the molecular-replacement program Phaser (Read, 2001), with the paAzoR1 structure (PDB code 2v9c) as a model. The atomic model of Y131F_MRE was rebuilt and refined with Coot (Emsley & Cowtan, 2004), REFMAC 5.4 (Murshudov et al., 1997) and PHENIX (Adams et al., 2002). Initial translation, liberation and screw (TLS) parameters were determined with the program TLSMD (Painter & Merritt, 2006) and TLS refinement was performed with PHENIX (Adams et al., 2002). Noncrystallographic symmetry (NCS) restraints (tight main chain and moderate side chain) were applied to the two protein chains in the asymmetric unit. Water molecules were added with PHENIX (Adams et al., 2002), Coot (Emsley & Cowtan, 2004) and ARP/wARP (Morris et al., 2003). Model validation was performed with MolProbity (Davis et al., 2007) and multimer analysis was performed using PISA (Krissinel & Henrick, 2005). Data-collection and refinement statistics are shown in Table 1.",
    "LLM": "2. Materials and methods 2.1. Site-directed mutagenesis and transformation The pET28b(+) plasmid vector containing the sequence of paazor1 was isolated from a 6 ml culture of Escherichia coli BL21 (DE3) using a QIAprep Spin Miniprep Kit (Qiagen). Site-directed mutagenesis was achieved using a QuikChange II kit (Stratagene). The reaction mixture (50 ml), which contained 5 ml 10 reaction buffer, 50 ng pET28b(+) plasmid as template, 125 ng of both Y131F forward (50-CGTCGCCCAGTTCCGGCCGCTGC-30) and Y131F reverse (50-GCAGCGGCCGGAACTGGGCGACG-30) primers and 1 ml dNTP mix, was subjected to thermo-cycling: 30 s at 368 K and 12 cycles of 30 s at 368 K, 1 min at 328 K and 6 min at 341 K, followed by 2 min on ice. The reaction mixture was digested (10 U DpnI, 1 h, 310 K) to cleave all template DNA before being purified by gel electrophoresis in ethidium-bromide-stained agarose [1.0%(w/v)]. The sequence of the insert was confirmed and the pET28b(+)-Y131F plasmid vector was transformed into E. coli BL21 (DE3) pLysS as described previously (Wang et al., 2007). 2.2. Protein production, purification and characterization Like wild-type paAzoR1, the Y131F mutant was expressed with a hexahistidine tag; the protein was then purified via affinity purification as described previously (Wang et al., 2007). Spectra were recorded from 600 to 300 nm and the enzymic activities were determined as described previously (Wang et al., 2007). Thermostability was determined by incubating the enzyme (10 min) from 277 to 363 K followed by 1 min on ice before adding methyl red and NADPH and measuring the loss of absorbance as described in Wang et al. (2007). Initial velocities were also measured at different concentrations of substrate and cofactor [from 5 to 40 mM methyl red and from 0.1 to 2.0 mM NAD(P)H], while the concentration of the other substrate was kept constant (Wang et al., 2007). In each case, linearity was established. Apparent Km and Vmax values were obtained from Lineweaver\u2013Burk plots using the initial linear rates expressed in mM s1. 2.3. Protein crystallization and structural determination Crystals of the mutant enzyme in the presence of methyl red (Y131F_MRE) were grown by sitting-drop vapour diffusion. Protein drops were prepared by mixing equal volumes (1 ml) of protein solution (23 mg ml1 in sterile water with 2 mM methyl red) with mother liquor (Molecular Dimensions JCSG-plus screen condition E8; 0.1 M sodium acetate pH 4.5, 1.0 M diammonium hydrogen phosphate) in a 24-well CrystalPlate (Axygen Bioscience). Crystals were briefly transferred to a cryoprotectant solution consisting of 1:3 glycerol:mother liquor prior to freezing in liquid nitrogen. Data were collected on beamline ID14-EH4 at the European Synchrotron Radiation Facility (ESRF, Grenoble) using a Quantum 315 ADSC CCD detector. Data were integrated with iMOSFLM v.0.5.2 (Leslie, 1992) and scaled and merged with SCALA (Collaborative Computational Project, Number 4, 1994). The structure was solved using the molecular-replacement program Phaser (Read, 2001), with the paAzoR1 structure (PDB code 2v9c) as a model. The atomic model of Y131F_MRE was rebuilt and refined with Coot (Emsley & Cowtan, 2004), REFMAC 5.4 (Murshudov et al., 1997) and PHENIX (Adams et al., 2002). Initial translation, liberation and screw (TLS) parameters were determined with the program TLSMD (Painter & Merritt, 2006) and TLS refinement was performed with PHENIX (Adams et al., 2002). Noncrystallographic symmetry (NCS) restraints (tight main chain and moderate side chain) were applied to the two protein chains in the asymmetric unit. Water molecules were added with PHENIX (Adams et al., 2002), Coot (Emsley & Cowtan, 2004) and ARP/wARP (Morris et al., 2003). Model validation was performed with MolProbity (Davis et al., 2007) and multimer analysis was performed using PISA (Krissinel & Henrick, 2005). Data-collection and refinement statistics are shown in Table 1."
  },
  "28467413": {
    "bleu": 94.20451421201481,
    "meteor": 94.37187477118167,
    "rouge1_p": 0.9545255793616091,
    "rouge1_r": 0.9591388400702988,
    "rouge1_f": 0.9568266491343415,
    "rouge2_p": 0.9225721784776902,
    "rouge2_r": 0.927032967032967,
    "rouge2_f": 0.9247971935978951,
    "rougeL_p": 0.9523393091386095,
    "rougeL_r": 0.9569420035149385,
    "rougeL_f": 0.9546351084812624,
    "cosine_similarity": 0.8946964232168949,
    "precision": 0.8118279569892473,
    "recall": 0.8435754189944135,
    "ner_f1": 0.8273972602739725,
    "ner_tp": 151,
    "ner_fp": 35,
    "ner_fn": 28,
    "bertscore_p": 0.9657368063926697,
    "bertscore_r": 0.9707126021385193,
    "bertscore_f1": 0.968349039554596,
    "bertscore_scibert_error": "The expanded size of the tensor (3099) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 3099].  Tensor sizes: [1, 512]",
    "len_gt": 13430,
    "len_jl": 13400,
    "GT": "Material and methods Dyes and chemicals Highest purity reagent grade chemicals were used for the study. RR241, all bacterial growth mediums and bovine albumin serum (BSA) was purchased from Sigma-Aldrich, UK. Sodium chloride, and anhydrous Na\u2082SO\u2084 were purchased from Merck, Germany. The stock solution (100 mg/L) of reactive red 241 was prepared using deionized water and working solution obtained via serial dilution. No specific permission was required for any activity of the presented research. Isolation and selection of effective dye degrading strain Soil sample as described by Kalyani et al., [12] was collected from an abandoned site of a textile industry located in the suburbs of Lahore (coordinates of the area, DMS latitude 31\u02da 34\u00b4 55.3620\u02dd N; longitude 74\u02da 19\u00b4 45.7536\u02dd E) in March, 2016. About 1 g of soil was placed into M9 medium (10 mL) with RR241 (50 \u03bcg/mL) as sole carbon source and left in the dark at room temperature for about 2 weeks. About 1 mL of solution was removed after agitation and placed into fresh M9 medium amended with RR241 (50 \u03bcg/mL) and incubated for further 2 weeks. The process was repeated six times and finally dilutions were made and approx. 1 ml of enrichment culture was streaked onto nutrient agar plates (plates were made for each dilution) constituted with RR241 (50 \u03bcg/mL) and incubated overnight at 30\u02daC. Metabolizing colonies (showing decolourization) were isolated and maintained in M9 media plates followed by the generation of glycerol stocks (40%) from 16 hr culture of isolates individually in M9 media. To determine the decolourisation capacity and toxicity assessment, colonies with the clear zones were inoculated in the mineral salt medium (MSM) agar plates (K\u2082HPO\u2084 1.73 gL\u207b\u00b9; FeSO\u2084\u00b77H\u2082O 0.03 gL\u207b\u00b9; KH\u2082PO\u2084 0.68 gL\u207b\u00b9; peptone 1.0 gL\u207b\u00b9; MgSO\u2084\u00b77H\u2082O 0.1 gL\u207b\u00b9; NaCl 0.1 gL\u207b\u00b9; NH\u2084NO\u2083 1.0 gL\u207b\u00b9; CaCl\u2082\u00b72H\u2082O 0.02 gL\u207b\u00b9) with varied concentration of RR241 (0\u2013100 mg/L) at different pH (2\u201312). Bacterial strain with the maximum decolourisation ability was selected. The exponential phase bacterial cells (OD600 nm) of the isolates were harvested by centrifugation at 6000\u00d7g for 5 min at 4\u02daC to prepare the resting cells. These cells were then washed severally with MSM before storing at 4\u02daC. A 100 mL of Difco Sporulation medium (DSM) amended with RR241 (0\u2013200 mg/L) at a constant concentration of resting cells (100 \u03bcl/100 ml of media) was employed to analyse the degradation and decolourisation rate using a UV\u2013visible spectrophotometer at \u03bbmax = 541. For this purpose, 2 mL of the aliquot was withdrawn from the culture media at regular intervals of 0\u201348 h and the decolourisation percentage (% D) calculated as follows: % \ud835\udc37 = \ud835\udc3c \ud835\udc5b \ud835\udc56 \ud835\udc61 \ud835\udc56 \ud835\udc4e \ud835\udc59 \ud835\udc4e \ud835\udc4f \ud835\udc60 \u2212 \ud835\udc39 \ud835\udc56 \ud835\udc5b \ud835\udc4e \ud835\udc59 \ud835\udc4e \ud835\udc4f \ud835\udc60 \ud835\udc3c \ud835\udc5b \ud835\udc56 \ud835\udc61 \ud835\udc56 \ud835\udc4e \ud835\udc59 \ud835\udc4e \ud835\udc4f \ud835\udc60 \u00d7 100 %D= Initialabs Initialabs\u2212Finalabs \u200b \u00d7100 Morphological and phylogenetic characterisation of bacterial strain The most efficient strain (S1) exhibiting the highest decolourisation potential was analysed for its morphology and physico-biochemical properties. The estimations of colony shape, size and gram staining were carried out initially. Further tests such as catalase, oxidase, urease, haemolytic activity, motility, indole, hydrolysis of indole and DNase were performed following standard procedures [13] to give a complete physico-chemical evaluation of the isolates. These tests were then evaluated according to Bergey\u2019s manual [14] to estimate the physico-chemical properties of the strain. Final identification was conducted through 16S rRNA sequence determination. For this overnight culture (of isolated bacteria) was centrifuged (13000 g for 5 min) and the resulting pellet was washed thrice with milliQ water (\u00d71) with final re-suspension was accomplished in 15 \u03bcl milliQ water followed by boiling at 95\u02daC for 5 min. Sample was cooled (on ice), spun (13000 g) for 5 min and finally the supernatant was collected. Gene was then amplified by PCR using dNTPs (200 \u03bcM each), GoTaq DNA polymerase, PCR buffer soln. (Promega) and primers AMP_F (5\u00b4: GAG AGT TTG ATY CTG GCT CAG; Tm = 60.5) and AMP_R (5\u00b4: AAG GAG GTG ATC CAR CCG CA; Tm = 68.9). PCR cycle consisted of initial denaturation at 95\u02daC for 2 min (1 cycle); denaturation at 95\u02daC for 30 s (30 cycles); annealing at 55\u02daC for 30 s (20 cycles); extension at 72\u02daC for 1 min 30 s (20 cycles); final extension at 72\u02daC for 1 min (1 cycle). The amplicon (PCR product; 1500 bp) was analysed on 1% agarose gel and purified by PCR purification kit (QIAquick; Qiagen). PCR product was quantified by a Nanodrop 2000 spectrometer (Thermo scientific) and sequenced with both the selected primers (AMP_F and AMP_R; Macrogen Inc.). Seqman was employed to assemble the sequences and create a contig, which was searched on RDP database (https://rdp.cme.msu.edu ). After custom sequencing by Macrogen Inc. the quality-trimmed extended contigs were generated by SPAdes programme, 3.6.1vs [15]. After removing gaps and analysing highly variable positions, a phylogenetic tree was generated from these contigs using Phylogeny.fr, 2016. Phylogenetic analysis The DNA extracted from the harvested bacterial biomass (isolated strain) and its 16s rDNA was amplified and sequenced by universal forward primer: 5\u2019-AGA GTT TGA TCC TGG TCA GAA CGC T-3\u2019 and reverse primer: 5\u2019-TAC GGC TAC CTT GTT ACG ACT TCA CCC C-3\u2019. After removing gaps and analysing highly variable positions a phylogenetic tree was generated. Extraction of enzyme and gel electrophoresis The resting cells from strain S1 were grown at 30\u02daC in liquid MSM medium for 24 hrs to obtain a mid-log phase. About 10 ml of sample was taken from it and centrifuged for 15 min at 5000 rpm. Supernatant thus obtained was filtered by a sterile syringe filter (0.45 mm, Millipore). This supernatant was designated to be extracellular protein and was used against different concentration of RR241 to determine the degradation rate and products. To determine the intracellular activity of azoreductase, the pellet, thus obtained (after centrifugation) was sonicated (Sonics vibracell ultrasonic processor). In order to breakdown the cell wall and release the cytosolic proteins, the sonicator was set at 40 amps with 8 strokes per 4 seconds. The sonication was done for 2 min and the sample was cooled on ice before centrifugation. Sonicated sample was centrifuged (12,000\u00d7g, 20 min) and the supernatant containing crude enzyme was transferred to a fresh tube. The crude enzyme was then precipitated by addition of ammonium sulphate (80% saturation, 4\u02daC). Precipitated enzyme was separated from the ammonium sulphate solution in the form of a pellet by centrifugation (10,000\u00d7g, 20 min). The pellet was further dissolved and dialyzed against 0.1 M phosphate buffer (pH 7.2) for 24 hrs at 4\u02daC to remove low molecular weight impurities. The contents of the dialysis tubing were transferred to a tube, lyophilized and kept frozen at -80\u02daC till further use. The lyophilized enzyme was re-dissolved in 0.1 M phosphate buffer (pH 7.2) and applied to a Sephadex G-100 column (1 cm \u00d7 30 cm, Amersham Biosciences), previously equilibrated with the same buffer. The enzyme was eluted following linear gradient of 0\u20130.5 M with phosphate buffer saline (pH 7.2, 0.5 M NaCl). About 2 ml fractions were collected at a flow rate of 6 ml/hr. Active fractions, thus obtained were pooled and lyophilized and was used as pure enzyme in further assays. The molecular mass of the protein was determined through polyacrylamide gel electrophoresis (SDS-PAGE analysis) and concentration of protein was analysed through standard protein assay: Bradford assay with Bovine serum albumin (BSA, Sigma Aldrich). The molecular weight of the enzyme was determined on 10% gel containing 0.1% SDS through the electrophoretic mobility of the enzyme with reference to DNA ladder and bovine serum albumin (66 KDa). The homogeneity was determined by PAGE carried out on 10% acrylamide gel without SDS. To determine the monomeric nature, polyacrylamide gel was run under native condition. The gel was stained with 150 \u03bcM of Navitan fast blue already made up in 0.1 mM phosphate buffer (pH 7) and then transferred to NADH (0.25 mM) containing a phosphate buffer (pH 7) and incubated at 35\u02daC. A clear, colourless area against dye background predicted the activity in the gel. Enzyme assay The enzyme assay was determined by adding varying concentrations of RR241 in 1 ml cuvette placed at 1 cm path length in UV spectrometer. An amount of 100 \u03bcL of cell fraction, as prepared above, was added to each cuvette. Since the enzyme was rendered as NADH dependent, thus the reaction was initiated by the addition of 4\u20138 \u03bcL of NADH. A negative control was also run in parallel to the experiment with 0.1 g/mL of lysozyme in replacement of enzyme to determine the oxidative reaction of RR241. The results were calculated through the disappearance of colour determined through absorption at maximum wavelength (\u03bbmax = 541 nm). The results were recorded as a unit of enzyme activity, where one unit of enzyme activity was the enzyme required, under assay condition, to decolorize 1 \u03bcM of RR241 in one min. Localization of azoreductase activity To localise the activity of azoreductase, cell free extract and resting cells were used for intracellular activity, whereas cell debris and the cell free homogenate was used to determine extracellular activities. The resting cells were prepared by harvesting exponential-phase bacterial cells followed by centrifugation (6000 \u00d7 g at 4\u02daC for 5 min). After centrifugation, the supernatant (homogenate) was removed and the pellet (resting cells, approx. 100 \u03bcg wet wt.) was washed thrice with MS medium. The pellet was then transferred to a 250 ml flask with 100 ml nutrient solution and 50 mg/L of RR241. The pH of the medium was set to pH 7 and aliquots were taken at constant intervals for 48 hrs. The reduction in RR241 was evaluated through determining wavelength at \u03bb = 541 nm. A similar experiment was repeated for homogenate (50 ml homogenate to 100 ml nutrient solution; 50 mg/L RR241 at pH 7), cell-free extract (2 ml extract to 100 ml nutrient solution; 50 mg/L RR241 at pH 7) and cell debris (100 \u03bcg wet wt in similar reaction mixture as described before). The cell free extract was prepared by growing cells in MN medium for 3 hrs. The cells were harvested by centrifugation (6000 \u00d7 g at 4\u02daC for 5 min) and then sonicated (Sonics vibracell ultrasonic processor). The cell debris was again removed by centrifugation (6000 \u00d7 g at 4\u02daC for 5 min). The pellet was referred as cell debris and supernatant was used as a cell free extract. To determine NADH dependency, the reaction mixture contained about 20 mM potassium phosphate buffer (pH 7.2), 50 mg/L RR241, 0.5 mM of NADH and appropriate amount of reaction medium, i.e cell free extract, cell debris, homogenate or resting cells, so that the final volume is 2.5 ml. The reaction is stopped by boiling at 100\u02daC for 5 min followed by centrifugation. The reduction was determined by measuring wavelength at \u03bb = 541 nm. Degradation of RR241 enzyme The degradation products were evaluated by (HPLC) and Fourier Transformation Infra-Red spectroscopy (FTIR). For HPLC the supernatant, containing denatured enzyme (obtained through boiling the reaction mix), was extracted with an equal volume of ethyl acetate so that organic layer was separated from the dye component. The extract was then dried with anhydrous Na\u2082SO\u2084 and the final residues obtained were dissolved in 2 ml HPLC-grade methanol. The contents were then analyzed by HPLC, Waters 2690 instruments (Waters Corporation, UK), using C18 reversed phase column (symmetry 4.6\u2013250 mm). The mobile phase was 60% acetonitrile and 40% water at a flow rate of 0.5 ml/min. For FTIR, the cell free supernatant was spread evenly on NaCl tablet and washed thrice with acetone. The analysis was conducted out on Perkin-Elmer 2000 FTIR spectrometer in the mid IR region of 400\u20134000 cm\u207b\u00b9 with a resolution 4 cm\u207b\u00b9 at 100 scans speed. The derivatization of metabolites was performed on QP2010 Shimadzu Gas chromatography equipped with Mass Spectroscopy (GC-MS). The assembly had a built-in autosampler and the sample was separated over BD-5 column (30 m \u00d7 0.25 mm \u00d7 0.25 mm). Helium was used as a carrier gas at a flow rate of 1 mL/min. The sample was dissolved in HPLC grade acetonitrile (3 mg/mL) before injection into the port with help of microsyringe. The temperature of the injection port was kept at 250\u02daC, where the holding temperature was 100\u02daC and the detector temperature was kept to 260\u02daC. The full scan acquisition was employed to analyse the peaks and comparison was made with standard run as well as MS analysis. Phytotoxicity assay Phytotoxicity assay was performed to assess the toxicity of industrial effluent with RR241 before and after biodegradation by Staphylococcus sp. KU898286. To get this S. vulgare (wild type) and A. thaliana (Col 0) seeds (10 seeds each) were allowed to grow at under growth conditions of a photoperiod of 16/8 h (L/D), temperature 23\u00b12\u02daC, RH 50/65% in compost (Levington\u2019s F2 with sand and perlite; mixture of 4:1 organic compost) watered with 500 mg/L of RR241 and biodegraded end product (recovered after completion of RR241 degradation by Staphylococcus sp. KU898286 by centrifugation and removal of cell debris). Germination (%), length of radicle and plumule was determined after 12 days of growth and evaluated against control seeds (watered with tap water).",
    "LLM": "Material and methods Dyes and chemicals Highest purity reagent grade chemicals were used for the study. RR241, all bacterial growth mediums and bovine albumin serum (BSA) was purchased from Sigma-Aldrich, UK. Sodium chloride, and anhydrous Na2SO4 were purchased from Merck, Germany. The stock solution (100mg/L) of reactive red 241 was prepared using deionized water and working solution obtained via serial dilution. No specific permission was required for any activity of the presented research. Isolation and selection of effective dye degrading strain Soil sample as described by Kalyani et al., [12] was collected from an abandoned site of a textile industry located in the suburbs of Lahore (coordinates of the area, DMS latitude 31\u02da 34\u00b4 55.3620\u02dd N; longitude 74\u02da 19\u00b4 45.7536\u02dd E) in March, 2016. About 1g of of soil was placed into M9 medium (10mL) with RR241 (50\u03bcg/mL) as sole carbon source and left in the dark at room temperature for about 2 weeks. About 1 mL of solution was removed after agitation and placed into fresh M9 medium amended with RR241 (50\u03bcg/mL) and incubated for further 2 weeks. The process was repeated six times and finally dilutions were made and approx. 1ml of enrich\ufffe ment culture was streaked onto nutrient agar plates (plates were made for each dilution) con\ufffe stituted with RR241(50\u03bcg/mL) and incubated overnight at 30\u02daC. Metabolizing colonies (showing decolourization) were isolated and maintained in M9 media plates followed by the generation of glycerol stocks (40%) from 16hr culture of isolates individually in M9 media. To determine the decolourisation capacity and toxicity assessment, colonies with the clear zones were inoculated in the mineral salt medium (MSM) agar plates (K2HPO4 1.73 gL\u20131; FeSO4 7H2O 0.03 gL\u20131; KH2PO4 0.68 gL\u20131; peptone 1.0 gL\u20131; MgSO4\u00017H2O 0.1 gL\u20131; NaCl 0.1 gL\u20131; NH4NO3 1.0 gL\u20131; CaCl2\u00012H2O 0.02 gL\u20131) with varied concentration of RR241 (0\u2013100 mg/L) at different pH (2\u201312). Bacterial strain with the maximum decolourisation ability was selected. The exponential phase bacterial cells (OD600nm) of the isolates were harvested by centrifu\ufffe gation at 6000\u00d7g for 5 min at 4\u02daC to prepare the resting cells. These cells were then washed severally with MSM before storing at 4\u02daC. A 100 mL of Difco Sporulation medium (DSM) amended with RR241 (0 \u2013 200mg/L) at a constant concentration of resting cells (100\u03bcl/100ml of media) was employed to analyse the degradation and decolourisation rate using a UV\u2013visi\ufffe ble spectrophotometer at \u03bbmax = 541. For this purpose, 2 mL of the aliquot was withdrawn from the culture media at regular intervals of 0\u201348 h and the decolourisation percentage (% D) calculated as follows: % D \u00bc Initial abs: \ufffe Final abs: Initial abs: 100 \u00f01\u00de Morphological and phylogenetic characterisation of bacterial strain The most efficient strain (S1) exhibiting the highest decolourisation potential was analysed for its morphology and physico-biochemical properties. The estimations of colony shape, size and gram staining were carried out initially. Further tests such as catalase, oxidase, urease, haemo\ufffe lytic activity, motility, indole, hydrolysis of indole and DNase were performed following stan\ufffe dard procedures [13] to give a complete physico-chemical evaluation of the isolates. These tests were then evaluated according to Bergey\u2019s manual [14] to estimate the physico-chemical properties of the strain. Final identification was conducted through 16S rRNA sequence deter\ufffe mination. For this overnight culture (of isolated bacteria) was centrifuged (13000g for 5min) and the resulting pellet was washed thrice with milliQ water (\u00d71) with final re-suspension was accomplished in 15\u03bcl milliQ water followed by boiling at 95\u02daC for 5 min. Sample was cooled (on ice), spun (13000g) for 5 min and finally the supernatant was collected. Gene was then amplified by PCR using dNTPs (200\u03bcM each), GoTaq DNA polymerase, PCR buffer soln. (Promega) and primers AMP_F (5': GAG AGT TTG ATY CTG GCT CAG; Tm = 60.5) and AMP_R (5': AAG GAG GTG ATC CAR CCG CA; Tm = 68.9). PCR cycle consisted of initial denaturation at 95\u02daC for 2 min (1 cycle); denaturation at 95\u02daC for 30 s (30 cycles); annealing at 55\u02daC for 30 s (20 cycles); extension at 72\u02daC for 1 min 30 s (20 cycles); final extension at 72\u02daC for 1 min (1 cycle). The amplicon (PCR product; 1500 bp) was analysed on 1% agarose gel and purified by PCr purification kit (QIAquick; Qiagen). PCR product was quanitified by a Nandrop 2000 spectrometer (Thermo scientific) and sequenced with both the selected primers (AMP_F and AMP_R; Macrogen Inc.). Seqman was employed to esemble the sequences and create a contig, which was searched on RDP database (https://rdp.cme.msu. edu). After custom sequencing by Macrigen Inc. the quality-trimmed extended contigs were generated by SPAdes programme, 3.6.1vs [15]. After removing gaps and analysing highly variable positions, a phylogenetic tree was generated from these contigs using Phylogeny.fr, 2016. Phylogenetic analysis The DNA extracted from the harvested bacterial biomass (isolated strain) and its 16s rDNA was amplified and sequenced by universal forward primer: 5\u2019-AGA GTT TGA TCC TGG TCA GAA CGC T-3\u2019 and reverse primer \u20185-TAC GGC TAC CTT GTT ACG ACT TCA CCC C-3\u2019. After removing gaps and analysing highly variable positions a phylogenetic tree was generated. Extraction of enzyme and gel electrophoresis The resting cells from strain S1 were grown at 30\u02daC in liquid MSM medium for 24 hrs to obtain a mid-log phase. About 10 ml of sample was taken from it and centrifuged for 15 min at 5000 rpm. Supernatant thus obtained was filtered by a sterile syringe filter (0.45 mm, Milli\ufffe pore). This supernatant was designated to be extracellular protein and was used against differ\ufffe ent concentration of RR241 to determine the degradation rate and products. To determine the intracellular activity of azoreductase, the pellet, thus obtained (after centrifugation) was soni\ufffe cated (Sonics vibracell ultrasonic processor). In order to breakdown the cell wall and release the cytosolic proteins, the sonicator was set at 40 amps with 8 strokes per 4 seconds. The soni\ufffe cation was done for 2 min and the sample was cooled on ice before centrifugation. Sonicated sample was centrifuged (12,000\u00d7g, 20 min) and the supernatant containing crude enzyme was transferred to a fresh tube. The crude enzyme was then precipitated by addition of ammonium sulphate (80% saturation, 4\u02daC). Precipitated enzyme was separated from the ammonium sul\ufffe phate solution in the form of a pellet by centrifugation (10,000\u00d7g, 20 min). The pellet was fur\ufffe ther dissolved and dialyzed against 0.1 M phosphate buffer (pH 7.2) for 24 hrs at 4\u02daC to remove low molecular weight impurities. The contents of the dialysis tubing were transferred to a tube, lyophyilized and kept frozen at -80\u02daC till further use. The lyophilized enzyme was re-dissolved in 0.1 M phosphate buffer (pH 7.2) and applied to a sephadex G-100 column (1cm \u00d7 30 cm, Amersham Biosciences), previously equilibrated with the same buffer. The enzyme was eluted following linear gradient of 0\u20130.5 M with phosphate buffer saline (pH7.2, 0.5 M NaCl). About 2ml fractions were collected at a flow rate of 6 ml/ hr. Active fractions, thus obtained were pooled and lyophilized and was used as pure enzyme in further assays. The molecular mass of the protein was determined through polyacrylamide gel electrophoresis (SDS-PAGE analysis) and concentration of protein was analysed through standard protein assay: Bradford assay with Bovin albumin serum (BSA, Sigma Aldrich). The molecular weight of the enzyme was deter\ufffe mined on 10% gel containing 0.1% SDS through the electrophoretic mobility of the enzyme with reference to DNA ladder and bovin serum albumin (66 KDa). The homogeneity was deter\ufffe mined by PAGE carried out on 10% acrylamide gel without SDS. To determine the monomeric nature, polyacrylamide gel was run under native condition. The gel was stained with 150 \u03bcM of Navitan fast blue already made up in 0.1 mM phosphate buffer (pH 7) and then transferred to NADH (0.25mM) containing a phosphate buffer (pH 7) and incubated at 35\u02daC. A clear, colour\ufffe less area against dye background predicted the activity in the gel. Enzyme assay The enzyme assay was determined by adding varying concentrations of RR241 in 1 ml cuvette placed at 1 cm path length in UV spectrometer. An amount of 100 \u03bcL of cell fraction, as pre\ufffe pared above, was added to each cuvette. Since the enzyme was rendered as NADH dependent, thus the reaction was initiated by the addition of 4\u20138 \u03bcL of NADH. A negative control was also run in parallel to the experiment with 0.1g/mL of lysozyme in replacement of enzyme to deter\ufffe mine the oxidative reaction of RR241. The results were calculated through the disappearance of colour determined through absorption at maximum wavelength (\u03bbmax = 541 nm). The results were recorded as a unit of enzyme activity, where one unit of enzyme activity was the enzyme required, under assay condition, to decolorize 1 \u03bcM of RR241 in one min. Localization of azoreductase activity To localise the activity of azoreductase, cell free extract and resting cells were used for intracel\ufffe lular activity, whereas cell debris and the cell free homogenate was used to determine extracel\ufffe lular activities. The resting cells were prepared by harvesting exponential-phase bacterial cells followed by centrifugation (6000 \u00d7 g at 4\u02daC for 5 min). After centrifugation, the supernatant (homogenate) was removed and the pellet (resting cells, approx. 100 \u03bcg wet wt.) was washed thrice with MS medium. The pellet was then transferred to a 250 ml flask with 100 ml nutrient solution and 50 mg/L of RR241. The pH of the medium was set to pH 7 and aliquots were taken at constant intervals for 48 hrs. The reduction in RR241 was evaluated through deter\ufffe mining wavelength at \u03bb = 541nm. A similar experiment was repeated for homogenate (50 ml homogenate to 100 ml nutrient solution; 50 mg/L RR241 at pH 7), cell-free extract (2 ml extract to 100 ml nutrient solution; 50 mg/L RR241 at pH 7) and cell debris (100 \u03bcg wet wt in similar reaction mixture as described before). The cell free extract was prepared by growing cells in MN medium for 3 hrs. The cells were harvested by centrifugation (6000 \u00d7 g at 4\u02daC for 5 min) and then sonicated (Sonics vibracell ultrasonic processor). The cell debris was again removed by centrifugation (6000 \u00d7 g at 4\u02daC for 5 min). The pellet was referred as cell debris and supernatant was used as a cell free extract. To determine NADH dependency, the reaction mixture contained about 20 mM potassium phosphate buffer (pH 7.2), 50 mg/L RR241, 0.5 mM of NADH and appropriate amount of reaction medium, i.e cell free extract, cell debris, homogenate or resting cells, so that the final volume is 2.5 ml. The reaction is stopped by boiling at 100\u02daC for 5 min followed by centrifuga\ufffe tion. The reduction was determined by measuring wavelength at \u03bb = 541nm. Degradation of RR241 enzyme The degradation products were evaluated by (HPLC) and Fourier Transformation Infra-Red spectroscopy (FTIR). For HPLC the supernatant, containing denatured enzyme (obtained through boiling the reaction mix), was extracted with an equal volume of ethyl acetate so that organic layer was separated from the dye component. The extract was then dried with anhy\ufffe drous Na2SO4 and the final residues obtained were dissolved in 2 ml HPLC-grade methanol. The contents were then analyzed by HPLC, Waters 2690 instruments (Waters Corporation, UK), using C18 reversed phase column (symmetry 4.6\u2013250 mm). The mobile phase was 60% acetonitrile and 40% water at a flow rate of 0.5 ml/min. For FTIR, the cell free supernatant was spread evenly on NaCl tablet and washed thrice with acetone. The analysis was conducted out on Perkin-Elmer 2000 FTIR spectrometer in the mid IR region of 400-4000cm-1 with a resolu\ufffe tion 4cm-1 at 100 scans speed. The derivatization of metabolites was performed on QP2010 Shimadzu Gas chromatography equipped with Mass Spectroscopy (GC-MS). The assembly had a built-in autosampler and the sample was separated over BD-5 column (30 m \u00d7 0.25 mm \u00d7 0.25 mm). Helium was used as a carrier gas at a flow rate of 1 mL/min. The sample was dissolved in HPLC grade acetonitrile (3 mg/ mL) before injection into the port with help of microsyringe. The temperature of the injection port was kept at 250\u02daC, where the holding tem\ufffe perature was 100\u02daC and the detector temperature was kept to 260\u02daC. The full scan acquisition was employed to analyse the peaks and comparison was made with standard run as well as MS analysis. Phytotoxicity assay Phytotoxicity assay was performed to assess the toxicity of industrial effluent with RR241 before and after biodegradation by Staphylococcus sp. KU898286. To get this S. vulgare (wild type) and A. thaliana (Col 0) seeds (10 seeds each) were allowed to grow at under growth con\ufffe ditions of a photoperiod of 16/8h (L/D), temperature 23\u00b12\u02daC, RH 50/65% in compost (Leving\ufffe ton\u2019s F2 with sand and perlite; mixture of 4:1 organic compost) watered with 500mg/L of RR241 and biodegraded end product (recovered after completion of RR241 degradation by Staphylococcus sp. KU898286 by centrifugation and removal of cell debris). Germination (%), length of radicle and plumule was determined after 12 days of growth and evaluated against control seeds (watered with tap water)."
  },
  "26724685": {
    "bleu": 95.28043775649795,
    "meteor": 94.96023013655108,
    "rouge1_p": 0.9613636363636363,
    "rouge1_r": 0.9694423223834988,
    "rouge1_f": 0.9653860783567896,
    "rouge2_p": 0.9378316906747536,
    "rouge2_r": 0.9457186544342507,
    "rouge2_f": 0.9417586600685192,
    "rougeL_p": 0.9613636363636363,
    "rougeL_r": 0.9694423223834988,
    "rougeL_f": 0.9653860783567896,
    "cosine_similarity": 0.9050440033743179,
    "precision": 0.9024390243902439,
    "recall": 0.9098360655737705,
    "ner_f1": 0.9061224489795918,
    "ner_tp": 111,
    "ner_fp": 12,
    "ner_fn": 11,
    "bertscore_p": 0.9404511451721191,
    "bertscore_r": 0.9662646055221558,
    "bertscore_f1": 0.9534617066383362,
    "bertscore_scibert_error": "The expanded size of the tensor (1869) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1869].  Tensor sizes: [1, 512]",
    "len_gt": 7760,
    "len_jl": 7768,
    "GT": "2. Materials and method 2.1. Chemicals, bacterial strains and plasmids Eight commercially used textile azo dyes including remazol black B, methyl red, maxilon blue, sulphonyl scarlet BNLE, sulphonyl blue TLE, sulphonyl green BLE, remazol black N, and entrazol blue IBC were purchased from Ciba Geigy (GmbH, Germany). Isopropyl-\u03b2-D-thiogalactopyranoside (IPTG) was from Sigma Aldrich (St. Louis, MO, USA). PCR kit was obtained from Kawsar Biotech Company (Iran). Other molecular biology kits, restriction endonucleases and T4 DNA ligase were from Fermentas (Germany). Unless stated differently, all other chemicals were obtained from Merck (Darmstadt, Germany). All chemicals were of analytical grade purity. E. coli DH5\u03b1 cells, E. coli BL21 (DE3) cells and pET21a vector were purchased from Novagen (USA). H. elongata was obtained from Iranian Biological Resource Center (IBRC). 2.2. Culture conditions for decolorization The decolorization experiments were carried out in culture tubes containing 10 mL of basic decolorization medium (glucose, 1% w/v; yeast extract, 0.5% w/v; NaCl, 5% w/v and azo dye, 0.005% w/v). The pH was adjusted to 7.2 with 1 M KOH before sterilization. Glucose and azo dye stock solutions were prepared and autoclaved separately at 121 \u00b0C for 15 min. The sterilized medium in culture tubes was inoculated with 1% of 1.5 \u00d7 10^8 CFU mL\u22121 of each of the tested bacterial suspensions (H. elongata, E. coli BL21 (DE3) with or without AzoH) and incubated at 34 \u00b0C (in the case of H. elongata) or 37 \u00b0C (in the case of E. coli strains) in the anoxic condition (no shaking) up to 5 days depending on the decolorization completion. The effect of various parameters including pH (5\u201311), temperature (25, 30, 35 and 40 \u00b0C), NaCl (1\u201325% w/v) and yeast extract (0.1\u20131% w/v) concentrations was also evaluated on the growth and decolorization ability of H. elongata towards remazol black B and methyl red. To measure decolorization extent, the absorbance of the clarified decolorization media was determined at \u03bb_max of the used dye. In all experiments, control reactions without bacteria were used to ensure that decolorization was only due to microbial activity and not because of the variation in parameters or light oxidation. 2.3. Recombinant DNA techniques Genomic DNA was extracted from H. elongata using phenol\u2013chloroform method [24]. The protein\u2013protein BLAST tool at the National Center for Biotechnology Information (NCBI) was used to perform homology searches. A search of the translated databases allowed the identification of the acp (acyl-carrier protein phosphodiesterase) gene in the whole genome sequence of H. elongata as the basis for the azoreductase activity. The putative DNA fragment encoding azoreductase gene, predicted based on the sequence homology in the whole genome of H. elongata (Genbank accession number FN869568.1), was amplified using PCR with the H. elongata genomic DNA as template and cloned in the pET21a vector which is characterized by a strong T7 promoter and a C-terminal 6 \u00d7 histidine tag. The forward primer 5\u2032-CGGGATCCATGACCACTCG-3\u2032 contained the BamHI restriction site upstream of the start codon, and the reverse primer 5\u2032-CCCAAGCTTGAGGCGTTCC-3\u2032 contained the HindIII site downstream of the gene. The PCR was performed using an Applied Biosystem 9700 thermal Cycler in 30 cycles: the first denaturation step at 94 \u00b0C for 3 min and each cycle consisting of denaturation at 94 \u00b0C for 1 min, annealing at 58 \u00b0C for 45 s, extension at 72 \u00b0C for 1 min and a final extension at 72 \u00b0C for 10 min. Size, yield and purity of the PCR product were considered according to the 1 kb DNA ladder (Takara, Otsu, Japan) in 0.8% agarose gel. The DNA fragment was purified by PCR purification kit. The purified fragment was digested with BamHI and HindIII and subsequently cloned in the pET21a expression vector that had been previously digested with the same enzymes. The ligation product was transformed into E. coli DH5\u03b1 cells. The recombinant plasmid was extracted using Fermentas plasmid mini-prep kit and transformed into the E. coli BL21 (DE3) expression cells. The azoreductase gene was sequenced from the pET21a using the T7 promoter and terminator primers (Novagen). Sequence analysis confirmed the correctness of the cloning procedure (Macrogen, Korea). 2.4. Protein expression and purification E. coli BL21 (DE3) cells containing recombinant pET21a plasmid were inoculated in 25 mL LB/Amp (100 \u03bcg mL\u22121) medium and incubated at 37 \u00b0C at 200 rpm. The overnight culture was inoculated into 200 mL LB broth medium (1% inoculation) containing ampicillin (100 \u03bcg mL\u22121) and incubated at 37 \u00b0C, 200 rpm. Upon reaching an OD600 = 0.5, the cells were induced for 3 h at 30 \u00b0C with Isopropyl-\u03b2-D-thiogalactopyranoside (IPTG), at a final concentration of 0.5 mM. The induced culture was centrifuged at 6000 \u00d7 g for 10 min at 4 \u00b0C to harvest the cells. The pellet was resuspended in 10 mL lysis buffer (50 mM sodium phosphate pH 7.6 and 1 mM phenylmethylsulfonyl fluoride (PMSF) in double-distilled water). The cells were disrupted by sonication using a SYCLON Ultra Sonic Cell SKL950-IIDN. Sonication was performed 10 times for 13 s ice. Cell debris was removed by centrifugation at 10000 \u00d7 g for 20 min at 4 \u00b0C. Ni^2+-affinity chromatography was used for the purification of hexa histidine-tagged recombinant protein. Therefore, the supernatant was loaded on Ni-NTA agarose column, equilibrated with sodium phosphate buffer 50 mM pH 7. Bound enzyme was eluted with 100 mM imidazole in elution buffer. Fractions of 1 mL were collected and those containing major enzyme activity were pooled. All protein purification steps were carried out at 4 \u00b0C. SDS\u2013PAGE (12.5%, w/v) was conducted to confirm the purity of the enzyme according to Laemmli method [25]. Protein markers (Novagen) were used as standards. The gel was stained using Coomassie brilliant blue R-250 (Bio-Rad). The enzyme concentration was determined by the Bradford assay using bovine serum albumin as standard [26]. 2.5. Enzyme activity and biochemical characterization The enzyme assay was carried out in a total reaction volume of 0.5 mL at room temperature. The reaction mixture contained 50 mM sodium phosphate buffer (pH 7), 200 \u03bcM methyl red and 0.03 mg/mL enzyme. The reaction was initiated by the addition of NADH prepared in 50 mM sodium phosphate buffer (pH 7) to a final concentration of 1 mM. Azoreductase activity was measured by the decrease in absorbance at 430 nm over a 1 min period using a PerkinElmer lambda 25 UV/VIS spectrophotometer. The extinction coefficient for methyl red at 430 nm was 23,360 M\u22121 cm\u22121. The enzyme activity was defined as the decrease in azo dye concentration (\u03bcM) per minute. All assays were performed in triplicates. The data were fitted to generate a Michaelis\u2013Menten equation, and apparent kinetic values were calculated. The potency of NADPH as an electron donor replacing NADH was checked. The enzyme activity towards other mentioned azo dyes was also tested. For this purpose, methyl red was replaced with each of the tested dyes in the reaction mix. However, quantitative parameters were only determined for methyl red and NADH. Enzyme-free control reactions were used in each case to assure that decolorization was only due to enzyme activity. 2.6. pH profile In order to find the optimal pH, azoreductase activity was measured towards methyl red in 50 mM mixed buffer of glycine, citrate and phosphate, within the pH range of 5\u201311. The enzyme concentration was fixed at 0.03 mg/mL. 2.7. NaCl profile Enzyme activity was examined towards methyl red in sodium phosphate buffer (50 mM) pH 7 at different concentrations of NaCl (0\u201325% w/v with 5% w/v interval) and a fixed enzyme concentration of 0.03 mg/mL.",
    "LLM": "2. Materials and method 2.1. Chemicals, bacterial strains and plasmids Eight commercially used textile azo dyes including rema\u00aczol black B, methyl red, maxilon blue, sulphonyl scarlet BNLE, sulphonyl blue TLE, sulphonyl green BLE, remazol black N, and entrazol blue IBC were purchased from Ciba Geigy (GmbH, Germany). Isopropyl--D-thiogalactopyranoside (IPTG) was from Sigma Aldrich (St. Louis, MO, USA). PCR kit was obtained from Kawsar Biotech Company (Iran). Other molecular biology kits, restriction endonucleases and T4 DNA ligase were from Fermen\u00actas (Germany). Unless stated differently, all other chemicals were obtained from Merck (Darmstadt, Germany). All chemicals were of analytical grade purity. E. coli DH5\u0004 cells, E. coli BL21 (DE3) cells andpET21a vector werepurchased fromNovagen(USA). H. elongata was obtained from Iranian Biological Resource Center (IBRC). 2.2. Culture conditions for decolorization The decolorization experiments were carried out in culture tubes containing 10 mL of basic decolorization medium (glucose, 1% w/v; yeast extract, 0.5% w/v; NaCl, 5% w/v and azo dye, 0.005% w/v). The pH was adjusted to 7.2 with 1 M KOH before sterilization. Glucose and azo dye stock solutions were prepared and autoclaved separately at 121 \u25e6C for 15 min. The sterilized medium in culture tubes was inoculated with 1% of 1.5 \u00d7 108 CFU mL\u22121 of each of the tested bacterial suspensions (H. elongata, E. coli BL21 (DE3) with or without AzoH) and incubated at 34 \u25e6C (in the case of H. elongata) or 37 \u25e6C (in the case of E. coli strains) in the anoxic condition (no shaking) up to 5 days depending on the decolorization completion. The effect of various parameters including pH (5\u201311), temper\u00acature (25, 30, 35 and 40 \u25e6C), NaCl (1\u201325% w/v) and yeast extract (0.1\u20131% w/v) concentrations was also evaluated on the growth and decolorization ability of H. elongata towards remazol black B and methyl red. To measure decolorization extent, the absorbance of the clarified decolorization media was determined at max of the used dye [18]. In all experiments, control reactions without bacteria were used to ensure that decolorization was only due to micro\u00acbial activity and not because of the variation in parameters or light oxidation. 2.3. Recombinant DNA techniques Genomic DNA was extracted from H. elongata using phenol\u00acchloroform method [24]. The protein\u2013protein BLAST tool at the National Center for Biotechnology Information (NCBI) was used to perform homology searches. A search of the translated databases allowed the identification of the acp (acyl-carrier protein phos\u00acphodiesterase) gene in the whole genome sequence of H. elongata as the basis for the azoreductase activity. The putative DNA frag\u00acment encoding azoreductase gene,predictedbasedonthe sequence homology in the whole genome of H. elongata (Genbank accession number FN869568.1), was amplified using PCR with the H. elon\u00acgata genomic DNA as template and cloned in the pET21a vector which is characterized by a strong T7 promoter and a C-terminal 6 \u00d7 histidine tag. The forward primer 5\u0004 -CGGGATCCATGACCACTCG -3\u0004 contained the BamHI restriction site upstream ofthe start codon, and the reverse primer 5\u0004 -CCCAAGCTTGAGGCGTTCC-3\u0004 contained the HindIII site downstream of the gene. The PCR was performed using an Applied Biosystem 9700 thermal Cycler in 30 cycles: the first denaturation step at 94 \u25e6C for 3 min and each cycle consisting of denaturation at 94 \u25e6C for 1 min, annealing at 58 \u25e6C for 45 s, exten\u00acsion at 72 \u25e6C for 1 min and a final extension at 72 \u25e6C for 10 min. Size, yield and purity of the PCR product were considered accord\u00acing to the 1 kb DNA ladder (Takara, Otsu, Japan) in 0.8% agarose gel. The DNA fragment was purified by PCR purification kit. The purified fragment was digested with BamHI and HindIII and sub\u00acsequently cloned in the pET21a expression vector that had been previously digested with the same enzymes. The ligation product was transformed into E. coli DH5\u0004 cells. The recombinant plasmid was extracted using Fermentas plasmid mini-prep kit and trans\u00acformedinto the E. coliBL21 (DE3) expressioncells. The azoreductase gene was sequenced from the pET21a using the T7 promoter and terminator primers (Novagen). Sequence analysis confirmed the correctness of the cloning procedure (Macrogen, Korea). 2.4. Protein expression and purification E. coli BL21 (DE3) cells containing recombinant pET21a plas\u00acmid were inoculated in 25 mL LB/Amp (100 g mL\u22121) medium and incubated at 37 \u25e6C at 200 rpm. The overnight culture was inoc\u00aculated into 200 mL LB broth medium (1% inoculation) containing ampicillin (100 g mL\u22121) and incubated at 37 \u25e6C, 200 rpm. Upon reaching an OD600 = 0.5, the cells were induced for 3 h at 30 \u25e6C with Isopropyl--D-thiogalactopyranoside (IPTG), at a final concentra\u00action of 0.5 mM. The induced culture was centrifuged at 6000 \u00d7 g for 10 min at 4 \u25e6C to harvest the cells. The pellet was resuspended in 10 mL lysis buffer (50 mM sodium phosphate pH 7.6 and 1 mM phenylmethylsulfonyl fluoride (PMSF) in double-distilled water)). The cells were disrupted by sonication using a SYCLON Ultra Sonic Cell SKL950-IIDN. Sonication was performed 10 times for 13 s ice. Cell debris was removed by centrifugation at 10000 \u00d7 g for 20 min at 4 \u25e6C. Ni2+-affinity chromatography was used for the purification of hexa histidine-tagged recombinant protein. Therefore, the super\u00acnatant was loaded on Ni-NTA agarose column, equilibrated with sodium phosphate buffer 50 mM pH 7. Bound enzyme was eluted with 100 mM imidazole in elution buffer. Fractions of 1 mL were collected and those containing major enzyme activity were pooled. All protein purification steps were carried out at 4 \u25e6C. SDS\u2013PAGE (12.5%, w/v) was conducted to confirm the purity of the enzyme according to Laemmli method [25]. Protein mark\u00acers (Novagen) were used as standards. The gel was stained using Coomassie brilliant blue R-250 (Bio-Rad). The enzyme concentra\u00action was determined by the Bradford assay using bovine serum albumin as standard [26]. 2.5. Enzyme activity and biochemical characterization The enzyme assay was carried out in a total reaction volume of 0.5 mL at room temperature. The reaction mixture contained 50 mM sodium phosphate buffer (pH 7), 200 M methyl red and 0.03 mg/mL enzyme. The reaction was initiated by the addition of NADH prepared in 50 mM sodium phosphate buffer (pH 7)to a final concentration of 1 mM. Azoreductase activity was measured by the decrease in absorbance at 430 nm over a 1 min period using a PerkinElmer lambda 25 UV/VIS spectrophotometer. The extinction coefficient for methyl red at 430 nm was 23,360 M\u22121 cm\u22121. The enzyme activ\u00acity was defined as the decrease in azo dye concentration (M) per minute. All assays were performed in triplicates. The data were fit\u00acted to generate a Michaelis\u2013Menten equation, and apparent kinetic values were calculated. The potency of NADPH as an electron donor replacing NADH was checked. The enzyme activity towards other mentioned azo dyes was also tested. For this purpose, methyl red was replaced with each of the tested dyes in the reaction mix. However, quanti\u00actative parameters were only determined for methyl red and NADH. Enzyme-free control reactions were used in each case to assure that decolorization was only due to enzyme activity. 2.6. pH profile In order to find the optimal pH, azoreductase activity was mea\u00acsured towards methyl red in 50 mM mixed buffer of glycine, citrate and phosphate, within the pH range of 5\u201311. The enzyme concen\u00actration was fixed at 0.03 mg/mL. 2.7. NaCl profile Enzyme activity was examined towards methyl red in sodium phosphate buffer (50 mM) pH 7 at different concentrations of NaCl (0\u201325% w/v with 5% w/v interval) and a fixed enzyme concentration of 0.03 mg/mL."
  },
  "22456237": {
    "bleu": 94.9115289058205,
    "meteor": 94.81292585937364,
    "rouge1_p": 0.9387990762124712,
    "rouge1_r": 0.9323394495412844,
    "rouge1_f": 0.9355581127733026,
    "rouge2_p": 0.8947976878612717,
    "rouge2_r": 0.8886337543053962,
    "rouge2_f": 0.8917050691244239,
    "rougeL_p": 0.9387990762124712,
    "rougeL_r": 0.9323394495412844,
    "rougeL_f": 0.9355581127733026,
    "cosine_similarity": 0.8653422853081143,
    "precision": 0.7976190476190477,
    "recall": 0.881578947368421,
    "ner_f1": 0.8375,
    "ner_tp": 67,
    "ner_fp": 17,
    "ner_fn": 9,
    "bertscore_p": 0.9336707592010498,
    "bertscore_r": 0.9296525716781616,
    "bertscore_f1": 0.9319345951080322,
    "bertscore_scibert_error": "The expanded size of the tensor (1220) must match the existing size (512) at non-singleton dimension 1.  Target sizes: [2, 1220].  Tensor sizes: [1, 512]",
    "len_gt": 4981,
    "len_jl": 4979,
    "GT": "2. Methods 2.1. Chemicals, bacterial strain and media Four Sudan dyes (Fig. S1) and other chemicals were all of analytical grade, purchased from Sigma\u2013Aldrich or TCI and used without further purification. S. oneidensis MR-1 (ATCC 700550) obtained from ATCC was routinely cultured in Luria\u2013Bertani (LB) broth medium aerobically at 30 \u00b0C. Sudan dye reduction studies were performed in modified M-R2A medium (Fries et al., 1994) containing (mg l\u207b\u00b9) KH\u2082PO\u2084, 250; K\u2082HPO\u2084, 400; KCl, 505; NH\u2084Cl, 800; CaCl\u2082\u00b72H\u2082O, 15; MgCl\u2082\u00b76H\u2082O, 20; FeSO\u2084\u00b77H\u2082O, 7; Na\u2082SO\u2084, 5; MnCl\u2082\u00b74H\u2082O, 5; H\u2083BO\u2083, 0.5; ZnCl\u2082, 0.5; CoCl\u2082\u00b76H\u2082O, 0.5; NiSO\u2084\u00b76H\u2082O, 0.5; and CuCl\u2082\u00b72H\u2082O, 0.3. Lactate (50 mM) was added as electron donor and the pH of the medium was adjusted to 7.0. 2.2. Removal of water-insoluble Sudan dyes by MR-1 cell suspensions The MR-1 cells cultured in a rotary incubator shaker (150 rpm) overnight were harvested by centrifugation (10,000g, 5 min) and washed twice with sterile phosphate buffered solution (20 mM, pH 7.0). Then the cell pellets were resuspended with M-R2A medium and held in the anaerobic chamber before use in the following studies. The experimental systems were 100 ml serum bottles containing 96 ml deoxygenated sterile medium and 10 mg l\u207b\u00b9 Sudan I. Addition of filter-sterilized sodium dodecyl sulfate (6.25 mg l\u207b\u00b9) and sonication were applied where needed to help disperse dye particles. MR-1 cells were added to the systems in anaerobic incubator at a final concentration of 0.7 g l\u207b\u00b9. After cell inoculation, samples were periodically taken with sterile needle and syringe and analyzed as described below. Control systems with heat-killed cells or without inoculation were also performed. All treatments and controls were run in triplicate. Factors affecting dye decolorization were systematically studied. Sudan I in experimental systems was replaced with 10 mg l\u207b\u00b9 each of Sudan II\u2013IV to study the removal capacities of S. oneidensis MR-1 for other Sudan dyes. To study the effects of electron donor concentration, 0.5\u201350 mM lactate were applied in experimental systems. Besides lactate, formate, acetate, pyruvate, citrate, salicylate, sucrose and glycerine were also added at 50 mM, respectively, to study the suitability of these organic substances to act as electron donor. To explore the optimal biomass, different amounts of MR-1 cells (0.15\u20131.0 g l\u207b\u00b9) were added into the systems. To study the effects of dye concentration, Sudan I concentration was ranged from 3 to 50 mg l\u207b\u00b9. In addition, 0.1 mM of 2-hydroxy-1,4-naphthoquinone (lawsone) or anthraquinone-2-sulfonate (AQS) were respectively added to the reduction systems to study the effects of quinone redox mediator on insoluble dye decolorization. 2.3. Preparation of cell extracts and enzyme assays Cells grown overnight were harvested by centrifugation (8000g, 25 \u00b0C) for 15 min and washed twice with sterile phosphate buffer (20 mM, pH 7.0). The pellets were frozen at \u201320 \u00b0C overnight. Cells thawed on ice and resuspended in sterile phosphate buffer (20 mM, pH 7.0) were disrupted by sonication (225 W, at 4 \u00b0C for 30 min). Then the homogenate was centrifuged at 60,000g for 20 min at 4 \u00b0C. The supernatant fraction was collected and used as cell extract whereas the pellets washed and resuspended in the phosphate buffer (20 mM, pH 7.0) was also saved for enzyme activity assay of membrane proteins. The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Azoreductase activity was tested by measuring the removal of Sudan I by different protein fractions. A 6 ml experimental system contained 5 ml cell extracts or pellets suspensions, 500 \u00b5L Sudan I (10 mg l\u207b\u00b9), and 500 \u00b5L NADH (100 \u00b5M). The reaction was initiated by adding NADH. Control systems contained no NADH, no protein fractions, or no Sudan I were also performed. Samples were taken at intervals to detect the disappearance of Sudan I. 2.4. Analytical methods After sampling in anaerobic incubator, equal volumes of ethanol (for Sudan I) or acetonitrile (for Sudan II\u2013IV) were added to solubilize samples, which were then put in boiled water-bath for 20 min to remove the color influence of cytochromes of S. oneidensis MR-1. Then the samples were centrifuged at 10,000g for 5 min to remove cell debris and absorbance of the supernatant was measured at 487, 503, 527 and 521 nm for Sudan I\u2013IV, respectively. Linear relationship was found between absorbance values and dye concentrations (Fig. S2). The removal of Sudan I and generation of products were also analyzed with high performance liquid chromatography (HPLC, Shimadzu LC-20AT, Japan) with a C18 column (Hypersil ODS-2, 5 \u00b5m, 4.6 \u00d7 250 mm) and UV-detector (detection wavelengths of 500 and 280 nm) after dissolving samples in ethanol or acetonitrile. The mobile phase was a mixture of methanol and ultra pure water (70:30, v:v) or a linear gradient of methanol from 50% to 80% in ultra-pure water at a flow rate of 1.0 ml min\u207b\u00b9.",
    "LLM": "2. Methods 2.1. Chemicals, bacterial strain and media Four Sudan dyes (Fig. S1) and other chemicals were all of ana\ufffelytical grade, purchased from Sigma\u2013Aldrich or TCI and used with\ufffeout further purification. S. oneidensis MR-1 (ATCC 700550) obtained from ATCC was routinely cultured in Luria\u2013Bertani (LB) broth medium aerobically at 30 C. Sudan dye reduction studies were performed in modified M-R2A medium (Fries et al., 1994) containing (mg l1 ) KH2PO4, 250; K2HPO4, 400; KCl, 505; NH4Cl, 800; CaCl22H2O, 15; MgCl26H2O, 20; FeSO47H2O, 7; Na2SO4, 5; MnCl24H2O, 5; H3BO3, 0.5; ZnCl2, 0.5; CoCl26H2O, 0.5; Ni\ufffeSO46H2O, 0.5; and CuCl22H2O, 0.3. Lactate (50 mM) was added as electron donor and the pH of the medium was adjusted to 7.0. 2.2. Removal of water-insoluble Sudan dyes by MR-1 cell suspensions The MR-1 cells cultured in a rotary incubator shaker (150 rpm) overnight were harvested by centrifugation (10,000g, 5 min) and washed twice with sterile phosphate buffered solution (20 mM, pH 7.0). Then the cell pellets were resuspended with M-R2A med\ufffeium and held in the anaerobic chamber before use in the following studies. The experimental systems were 100 ml serum bottles contain\ufffeing 96 ml deoxygenated sterile medium and 10 mg l1 Sudan I. Addition of filter-sterilized sodium dodecyl sulfate (6.25 mg l1 ) and sonication were applied where needed to help disperse dye particles. MR-1 cells were added to the systems in anaerobic incu\ufffebator at a final concentration of 0.7 g l1 . After cell inoculation, samples were periodically taken with sterile needle and syringe and analyzed as described below. Control systems with heat-killed cells or without inoculation were also performed. All treatments and controls were run in triplicate. Factors affecting dye decolorization were systematically stud\ufffeied. Sudan I in experimental systems was replaced with 10 mg l1 each of Sudan II\u2013IV to study the removal capacities of S. oneidensis MR-1 for other Sudan dyes. To study the effects of electron donor concentration, 0.5\u201350 mM lactate were applied in experimental systems. Besides lactate, formate, acetate, pyruvate, citrate, salicy\ufffelate, sucrose and glycerine were also added at 50 mM, respectively, to study the suitability of these organic substances to act as elec\ufffetron donor. To explore the optimal biomass, different amounts of MR-1 cells (0.15\u20131.0 g l1 ) were added into the systems. To study the effects of dye concentration, Sudan I concentration was ranged from 3 to 50 mg l1 . In addition, 0.1 mM of 2-hydroxy-1,4-naph\ufffethoquinone (lawsone) or anthraquinone-2-sulfonate (AQS) were respectively added to the reduction systems to study the effects of quinone redox mediator on insoluble dye decolorization. 2.3. Preparation of cell extracts and enzyme assays Cells grown overnight were harvested by centrifugation (8000g, 25 C) for 15 min and washed twice with sterile phosphate buffer (20 mM, pH 7.0). The pellets were frozen at 20 C overnight. Cells thawed on ice and resuspended in sterile phosphate buffer (20 mM, pH 7.0) were disrupted by sonication (225 W, at 4 C for 30 min). Then the homogenate was centrifuged at 60,000g for 20 min at 4 C. The supernatant fraction was collected and used as cell extract whereas the pellets washed and resuspended in the phosphate buffer (20 mM, pH 7.0) was also saved for enzyme activity assay of membrane proteins. The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Azoreductase activity was tested by measuring the removal of Sudan I by different protein fractions. A 6 ml experimental system contained 5 ml cell extracts or pellets suspensions, 500 lL Sudan I (10 mg l1 ), and 500 lL NADH (100 lM). The reaction was initiated by adding NADH. Control systems contained no NADH, no protein fractions, or no Sudan I were also performed. Samples were taken at intervals to detect the disappearance of Sudan I. 2.4. Analytical methods After sampling in anaerobic incubator, equal volumes of ethanol (for Sudan I) or acetonitrile (for Sudan IIIV) were added to solu\ufffebilize samples, which were then put in boiled water-bath for 20 min to remove the color influence of cytochromes of S. oneiden\ufffesis MR-1. Then the samples were centrifuged at 10,000g for 5 min to remove cell debris and absorbance of the supernatant was mea\ufffesured at 487, 503, 527 and 521 nm for Sudan I\u2013IV, respectively. Linear relationship was found between absorbance values and dye concentrations (Fig. S2). The removal of Sudan I and generation of products were also analyzed with high performance liquid chromatography (HPLC, Shimadzu LC-20AT, Japan) with a C18 column (Hypersil ODS-2, 5 lm, 4.6 \u0004 250 mm) and UV-detector (detection wavelengths of 500 and 280 nm) after dissolving samples in ethanol or acetoni\ufffetrile. The mobile phase was a mixture of methanol and ultra pure water (70:30, v:v) or a linear gradient of methanol from 50% to 80% in ultra-pure water at a flow rate of 1.0 ml min1 ."
  }
}