PMC:3395577 / 21375-29151 JSONTXT

{"project":"MicrobeTaxon","denotations":[{"id":"T48","span":{"begin":23,"end":32},"obj":"2"},{"id":"T49","span":{"begin":65,"end":116},"obj":"1517"},{"id":"T50","span":{"begin":228,"end":244},"obj":"562"},{"id":"T51","span":{"begin":1096,"end":1128},"obj":"1517"},{"id":"T52","span":{"begin":1679,"end":1686},"obj":"562"},{"id":"T53","span":{"begin":1982,"end":1989},"obj":"562"},{"id":"T54","span":{"begin":5764,"end":5771},"obj":"562"}],"namespaces":[{"prefix":"_base","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"text":"Materials and Methods\n\nBacterial Strains, Plasmids, Growth Media\nThermoanaerobacterium thermosaccharolyticum DSM 571 was purchased from DSMZ (http://www.dsmz.de). It was grown anaerobically at 60°C as described previously [17]. Escherichia coli JM109 and JM109(DE3) was grown at 37°C in Luria-Bertani medium (LB) and supplemented with ampicillin when required. The expression vectors pET-20b (Novagen) were employed as cloning vector and expression vector.\n\nDNA manipulation\nDNA was manipulated by standard procedures [25]. QIAGEN Plasmid Kit and QIAGEN MinElute Gel Extraction Kit (Qiagen, USA) were employed for the purification of plasmids and PCR products. DNA restriction and modification enzymes were purchased form TaKaRa (Dalian, China). DNA transformation was performed by electroporation using GenePulser (Bio-Rad, USA). Site-directed mutagenesis of genes and the modification of the plasmids were performed by inverse-PCR followed by phosporylation and self-ligation using T4 polynucleotide kinase and T4 DNA ligase.\n\nPlamid constructions\nThe β-glucosidase gene bgl was amplified from T. thermosaccharolyticum DSM 571 genomic DNA by PCR using primers bgl-1 and bgl-2 (Table 3), the PCR products were digested with Nde I and Xho I and inserted into pET-20b at Nde I and Xho I sites, yielding the plasmid pET-20-BGL.\nTable 3 Nucleotide sequences of used primers The boldface italic nucleotides represented mutations for optimizing codons. In order to improve the expression level of recombinant BGL, the internal region from 1st to 19th amino acids in open reading frame of bgl was mutated in situ by inverse-PCR to replace the rare codons with the optimal codons of E. coli; the primers for the inverse-PCR were designated as bgl-3 and bgl-4 (Table 3). Inverse-PCR with primers was carried out using Pyrobest with pET-20-BGL as template, generating the plasmid pET-20-BGLII.\n\nExpression and purification of BGL\nPlasmids pET-20-BG and pET-20-BGLII were transformed into E. coli JM109(DE3), and induced to expressed recombinant BGL by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to final concentration of 0.8 mM at OD600 about 0.7, and incubated further at 30°C for about 6 h.\nOne liters of the recombinant cells carrying pET-20-BGLII were harvested by centrifugation at 5,000 g for 10 min at 4°C, and washed twice with distilled water, resuspended in 50 mL of 5 mM imidazole, 0.5 mM NaCl, and 20 mM Tris–HCl buffer (pH 7.9), and French-pressured for three times. The cell extracts were heat treated (60°C, 30 min), and then cooled in an ice bath, and centrifuged (20,000 g, 4°C, 30 min). The resulting supernatants were loaded on to an immobilized metal affinity column (Novagen, USA), and eluded with 1 M imidazole, 0.5 M NaCl, and 20 mM Tris–HCl buffer (pH 7.9). Protein was examined by SDS-PAGE [26], and the protein bands were analyzed by density scanning with an image analysis system (Bio-Rad, USA). Protein concentration was determined by the Bradford method using BSA as a standard.\n\nDetermination of enzyme activities and properties\nThe reaction mixture, containing 50 mM imidole-potassium buffer (pH 6.4), 1 mM p-nitrophenyl-β-D-glucopyranoside, and certain amount of β-glucosidase in 0.2 mL, was incubated for 5 min at 70°C. The reaction was stopped by adding 1 mL of 1 M Na2CO3. The absorbance of the mixture was measured at 405 nm. One unit of enzyme activity was defined as the amount of enzyme necessary to liberate 1 μmol of pNP per min under the assay conditions.\nThe optimum pH for activity β-glucosidase was determined by incubation at 70°C for 5 min in the 50 mM imidole-potassium buffer from pH 4.8 to 8.4. The optimum temperature for the enzyme activity was determined by standard assay ranging from 45 to 85°C in the 50 mM imidole-potassium buffer, pH 6.0. The results were expressed as percentages of the activity obtained at either the optimum pH or the optimum temperature.\nThe pH stability of the enzyme was determined by measuring the remaining activity after incubating the enzyme (0.1 μg) at 50°C for 1 h in the 50 mM imidole-potassium buffer from pH 5.2 to 8.0. To determine the effect of temperature on the stability of BGL, the enzyme (0.1 μg) in the 50 mM imidole-potassium buffer (pH 6.4) was pre-incubated for various times at 50°C, 65°C, 68°C and 70°C in the absence of the substrate. The activity of the enzyme without pre-incubation was defined as 100%.\nThe effects of metals and chemical agents on β-glucosidase activity of purified enzyme (0.1 μg) were determined. Fe2+, Mg2+, Zn2+, Mn2+, Ca2+, K+, Al3+, Li2+, Cu2+, Co2+, and Hg2+ were assayed at concentrations of 1 mM in the reaction mixture. The chemical agents EDTA (10 mM) were assayed. The enzyme was incubated with each reagent for 10 min at 50°C before addition of p-nitrophenyl-β-D-glucopyranoside to initiate the enzyme reaction. Activity was determined as described above and was expressed as a percentage of the activity obtained in the absence of the chemical agents and metal cations.\nThe substrate specificity of the enzyme (0.1 μg) was tested by using following p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-L-arabinofuranoside, maltose, sucrose, and cellobiose. Kinetic constant of BGL was determined by measuring the initial rates at various p-nitrophenyl-β-D-glucopyranoside concentrations (0.2, 0.4, 0.6, 0.8, 1, 2, and 4.0 mM) or various cellobiose concentration (2, 4, 6, 8, 10, 12, 14, and 16 mM) under standard reaction conditions. The Ki value of glucose was defined as amount of glucose required for inhibiting 50% of the β-glucosidase activity and was given as the averages of three separate experiments performed in duplicate.\n\nPhylogenies analysis of BGL\nThe condon usage preference of E. coli in translation initiation region of pET-20-BGL was analyzed by using codon usage tool (http://gcua.schoedl.de/). The potential ORF of bgl was searched using the ORF search tool provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Database searching was performed with Blast at NCBI and against CAZy (http://www.cazy.org). The active site of the enzyme was analyzed with the prosite tool (http://prosite.expasy.org/scanprosite). The multiple sequence alignment tool Clustal X2.0 was used for multiple protein sequence alignment [27]. Sequences were further edited and aligned manually, when necessary, using the Mega 5 for editing. For phylogenetic analyses of conserved domains, sequences were trimmed so that only the relevant protein domains remained in the alignment [28]. Phylogenetic relationships were inferred using the Neighbor-Joining (NJ) and Maximum-Parsimony (MP) method as implemented in Paup 4.0 for the NJ and MP trees, the results were evaluated with 1000 bootstrap replicates [29]. The generated trees were displayed using TREEVIEW 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).\n\nAnalysis of cellobiose degradation\nThe cellobiose was treated with purified BGL, and the degradation was subjected to analysis of thin-layer chromatography (TLC) and HPLC. The reaction mixture (20 μL) contained 290 mM cellobiose, and 1 μg of BGL in 50 mM imidole-potassium buffer (pH 6.4). The reaction was performed for various times at 60°C, and stopped by heating for 5 min in a boiling water bath. After centrifuged for 10 min at 10,000 g, supernatants of the reaction mixtures were applied on silica gel TLC plates (60F254, Merck Co.). Sugars on the plates were partitioned with a solvent system consisting of n-butanol, acetic acid, and water (2:1:1, by vol/vol), and detected using the orcinol reagent [30]. The concentration of glucose was examined by HPLC on a carbohydrate analysis column (Waters Sugarpak1, USA) with water as a mobile phase.\n"}

{"project":"2_test","denotations":[{"id":"22571470-21464600-7770710","span":{"begin":223,"end":225},"obj":"21464600"},{"id":"22571470-5432063-7770711","span":{"begin":2817,"end":2819},"obj":"5432063"},{"id":"22571470-17846036-7770712","span":{"begin":6340,"end":6342},"obj":"17846036"},{"id":"22571470-21546353-7770713","span":{"begin":6583,"end":6585},"obj":"21546353"},{"id":"22571470-11298744-7770714","span":{"begin":7633,"end":7635},"obj":"11298744"}],"text":"Materials and Methods\n\nBacterial Strains, Plasmids, Growth Media\nThermoanaerobacterium thermosaccharolyticum DSM 571 was purchased from DSMZ (http://www.dsmz.de). It was grown anaerobically at 60°C as described previously [17]. Escherichia coli JM109 and JM109(DE3) was grown at 37°C in Luria-Bertani medium (LB) and supplemented with ampicillin when required. The expression vectors pET-20b (Novagen) were employed as cloning vector and expression vector.\n\nDNA manipulation\nDNA was manipulated by standard procedures [25]. QIAGEN Plasmid Kit and QIAGEN MinElute Gel Extraction Kit (Qiagen, USA) were employed for the purification of plasmids and PCR products. DNA restriction and modification enzymes were purchased form TaKaRa (Dalian, China). DNA transformation was performed by electroporation using GenePulser (Bio-Rad, USA). Site-directed mutagenesis of genes and the modification of the plasmids were performed by inverse-PCR followed by phosporylation and self-ligation using T4 polynucleotide kinase and T4 DNA ligase.\n\nPlamid constructions\nThe β-glucosidase gene bgl was amplified from T. thermosaccharolyticum DSM 571 genomic DNA by PCR using primers bgl-1 and bgl-2 (Table 3), the PCR products were digested with Nde I and Xho I and inserted into pET-20b at Nde I and Xho I sites, yielding the plasmid pET-20-BGL.\nTable 3 Nucleotide sequences of used primers The boldface italic nucleotides represented mutations for optimizing codons. In order to improve the expression level of recombinant BGL, the internal region from 1st to 19th amino acids in open reading frame of bgl was mutated in situ by inverse-PCR to replace the rare codons with the optimal codons of E. coli; the primers for the inverse-PCR were designated as bgl-3 and bgl-4 (Table 3). Inverse-PCR with primers was carried out using Pyrobest with pET-20-BGL as template, generating the plasmid pET-20-BGLII.\n\nExpression and purification of BGL\nPlasmids pET-20-BG and pET-20-BGLII were transformed into E. coli JM109(DE3), and induced to expressed recombinant BGL by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to final concentration of 0.8 mM at OD600 about 0.7, and incubated further at 30°C for about 6 h.\nOne liters of the recombinant cells carrying pET-20-BGLII were harvested by centrifugation at 5,000 g for 10 min at 4°C, and washed twice with distilled water, resuspended in 50 mL of 5 mM imidazole, 0.5 mM NaCl, and 20 mM Tris–HCl buffer (pH 7.9), and French-pressured for three times. The cell extracts were heat treated (60°C, 30 min), and then cooled in an ice bath, and centrifuged (20,000 g, 4°C, 30 min). The resulting supernatants were loaded on to an immobilized metal affinity column (Novagen, USA), and eluded with 1 M imidazole, 0.5 M NaCl, and 20 mM Tris–HCl buffer (pH 7.9). Protein was examined by SDS-PAGE [26], and the protein bands were analyzed by density scanning with an image analysis system (Bio-Rad, USA). Protein concentration was determined by the Bradford method using BSA as a standard.\n\nDetermination of enzyme activities and properties\nThe reaction mixture, containing 50 mM imidole-potassium buffer (pH 6.4), 1 mM p-nitrophenyl-β-D-glucopyranoside, and certain amount of β-glucosidase in 0.2 mL, was incubated for 5 min at 70°C. The reaction was stopped by adding 1 mL of 1 M Na2CO3. The absorbance of the mixture was measured at 405 nm. One unit of enzyme activity was defined as the amount of enzyme necessary to liberate 1 μmol of pNP per min under the assay conditions.\nThe optimum pH for activity β-glucosidase was determined by incubation at 70°C for 5 min in the 50 mM imidole-potassium buffer from pH 4.8 to 8.4. The optimum temperature for the enzyme activity was determined by standard assay ranging from 45 to 85°C in the 50 mM imidole-potassium buffer, pH 6.0. The results were expressed as percentages of the activity obtained at either the optimum pH or the optimum temperature.\nThe pH stability of the enzyme was determined by measuring the remaining activity after incubating the enzyme (0.1 μg) at 50°C for 1 h in the 50 mM imidole-potassium buffer from pH 5.2 to 8.0. To determine the effect of temperature on the stability of BGL, the enzyme (0.1 μg) in the 50 mM imidole-potassium buffer (pH 6.4) was pre-incubated for various times at 50°C, 65°C, 68°C and 70°C in the absence of the substrate. The activity of the enzyme without pre-incubation was defined as 100%.\nThe effects of metals and chemical agents on β-glucosidase activity of purified enzyme (0.1 μg) were determined. Fe2+, Mg2+, Zn2+, Mn2+, Ca2+, K+, Al3+, Li2+, Cu2+, Co2+, and Hg2+ were assayed at concentrations of 1 mM in the reaction mixture. The chemical agents EDTA (10 mM) were assayed. The enzyme was incubated with each reagent for 10 min at 50°C before addition of p-nitrophenyl-β-D-glucopyranoside to initiate the enzyme reaction. Activity was determined as described above and was expressed as a percentage of the activity obtained in the absence of the chemical agents and metal cations.\nThe substrate specificity of the enzyme (0.1 μg) was tested by using following p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-L-arabinofuranoside, maltose, sucrose, and cellobiose. Kinetic constant of BGL was determined by measuring the initial rates at various p-nitrophenyl-β-D-glucopyranoside concentrations (0.2, 0.4, 0.6, 0.8, 1, 2, and 4.0 mM) or various cellobiose concentration (2, 4, 6, 8, 10, 12, 14, and 16 mM) under standard reaction conditions. The Ki value of glucose was defined as amount of glucose required for inhibiting 50% of the β-glucosidase activity and was given as the averages of three separate experiments performed in duplicate.\n\nPhylogenies analysis of BGL\nThe condon usage preference of E. coli in translation initiation region of pET-20-BGL was analyzed by using codon usage tool (http://gcua.schoedl.de/). The potential ORF of bgl was searched using the ORF search tool provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). Database searching was performed with Blast at NCBI and against CAZy (http://www.cazy.org). The active site of the enzyme was analyzed with the prosite tool (http://prosite.expasy.org/scanprosite). The multiple sequence alignment tool Clustal X2.0 was used for multiple protein sequence alignment [27]. Sequences were further edited and aligned manually, when necessary, using the Mega 5 for editing. For phylogenetic analyses of conserved domains, sequences were trimmed so that only the relevant protein domains remained in the alignment [28]. Phylogenetic relationships were inferred using the Neighbor-Joining (NJ) and Maximum-Parsimony (MP) method as implemented in Paup 4.0 for the NJ and MP trees, the results were evaluated with 1000 bootstrap replicates [29]. The generated trees were displayed using TREEVIEW 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).\n\nAnalysis of cellobiose degradation\nThe cellobiose was treated with purified BGL, and the degradation was subjected to analysis of thin-layer chromatography (TLC) and HPLC. The reaction mixture (20 μL) contained 290 mM cellobiose, and 1 μg of BGL in 50 mM imidole-potassium buffer (pH 6.4). The reaction was performed for various times at 60°C, and stopped by heating for 5 min in a boiling water bath. After centrifuged for 10 min at 10,000 g, supernatants of the reaction mixtures were applied on silica gel TLC plates (60F254, Merck Co.). Sugars on the plates were partitioned with a solvent system consisting of n-butanol, acetic acid, and water (2:1:1, by vol/vol), and detected using the orcinol reagent [30]. The concentration of glucose was examined by HPLC on a carbohydrate analysis column (Waters Sugarpak1, USA) with water as a mobile phase.\n"}