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High phylogenetic diversity of glycosyl hydrolase family 10 and 11 xylanases in the sediment of Lake Dabusu in China.

Wang G, Huang X, Ng TB, Lin J, Ye XY - PLoS ONE (2014)

Bottom Line: A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences.Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi.These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.

View Article: PubMed Central - PubMed

Affiliation: College of Biological Science and Technology, Fuzhou University, Fuzhou 350108, P.R. China; National Engineering Laboratory for High-efficiency Enzyme Expression, Fuzhou 350002, P. R. China.

ABSTRACT
Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.

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pH and temperature activity profiles of purified recombinant XynAS10-66.A Effect of pH on XynAS10-66 activity. Activities at various pHs were assayed at 30°C in different buffer. B Effect of temperature on XynAS10-66 activity in Tris-HCl buffer (pH 9.0).
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pone-0112798-g004: pH and temperature activity profiles of purified recombinant XynAS10-66.A Effect of pH on XynAS10-66 activity. Activities at various pHs were assayed at 30°C in different buffer. B Effect of temperature on XynAS10-66 activity in Tris-HCl buffer (pH 9.0).

Mentions: The gene encoding the mature proteins was expressed in E. coli BL21 (DE3). Following induction with IPTG at 30°C for 12 h, substantial xylanase activity was detected in the culture supernatant of recombinant cells. Using beechwood xylan as the substrate, rXynAS10-66 showed the highest activity at pH 7.0, while it retained more than 95% activity at pH 9.0, and >60% of the maximum activity at pH 7.0–11.0 (Figure 4A). Using beechwood xylan as the substrate, the Km and Vmax values were 1.5±0.04 mg · mL−1, 1102±9.54 µmol · mg−1 · min−1, respectively. Although the optimum temperature of the recombinant XynAS10-66 is neutral, it has substantial activity at alkaline pH, with more than 80% and 60% activity being retained at pH 10 and pH 11, respectively. This characterization differs from those of alkaline xylanases obtained from Bacillus and Micrococcus sp. isolated from other soda lakes, which showed optimum activity at pH 8 to 9 [14], [15]. The optimal temperature for the enzyme activity of XynAS10-66 was 55°C (Figure 4B), while more than 60% activity was retained at 45°C to 65°C. The recombinant XynAS10-66 had the same optimum temperature as the alkaline xylanase from alkaliphilic Micrococcus sp. AR-135 [14]. However, it had a lower activity than that of xylanases from Bacillus halodurans S7 [13] and two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. [15]. BLAST analysis revealed that XynAS10-66 shared high similarity with a thermostable endo-beta-1,4-xylanase of Thermomonospora alba ULJB1 [41], which showed good activity at up to 95°C. Sequence alignment revealed that XynAS10-66 lacks a carbohydrate binding module (CBM) at the C terminal, which may have resulted in the lower temperature optimum of XynAS10-66. Future studies will be conducted to investigate this phenomenon. Moreover, other fragment sequences will be used to generate more full-length genes directly from the metagenomic DNA, and any genes generated will be characterized in subsequent studies.


High phylogenetic diversity of glycosyl hydrolase family 10 and 11 xylanases in the sediment of Lake Dabusu in China.

Wang G, Huang X, Ng TB, Lin J, Ye XY - PLoS ONE (2014)

pH and temperature activity profiles of purified recombinant XynAS10-66.A Effect of pH on XynAS10-66 activity. Activities at various pHs were assayed at 30°C in different buffer. B Effect of temperature on XynAS10-66 activity in Tris-HCl buffer (pH 9.0).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231106&req=5

pone-0112798-g004: pH and temperature activity profiles of purified recombinant XynAS10-66.A Effect of pH on XynAS10-66 activity. Activities at various pHs were assayed at 30°C in different buffer. B Effect of temperature on XynAS10-66 activity in Tris-HCl buffer (pH 9.0).
Mentions: The gene encoding the mature proteins was expressed in E. coli BL21 (DE3). Following induction with IPTG at 30°C for 12 h, substantial xylanase activity was detected in the culture supernatant of recombinant cells. Using beechwood xylan as the substrate, rXynAS10-66 showed the highest activity at pH 7.0, while it retained more than 95% activity at pH 9.0, and >60% of the maximum activity at pH 7.0–11.0 (Figure 4A). Using beechwood xylan as the substrate, the Km and Vmax values were 1.5±0.04 mg · mL−1, 1102±9.54 µmol · mg−1 · min−1, respectively. Although the optimum temperature of the recombinant XynAS10-66 is neutral, it has substantial activity at alkaline pH, with more than 80% and 60% activity being retained at pH 10 and pH 11, respectively. This characterization differs from those of alkaline xylanases obtained from Bacillus and Micrococcus sp. isolated from other soda lakes, which showed optimum activity at pH 8 to 9 [14], [15]. The optimal temperature for the enzyme activity of XynAS10-66 was 55°C (Figure 4B), while more than 60% activity was retained at 45°C to 65°C. The recombinant XynAS10-66 had the same optimum temperature as the alkaline xylanase from alkaliphilic Micrococcus sp. AR-135 [14]. However, it had a lower activity than that of xylanases from Bacillus halodurans S7 [13] and two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. [15]. BLAST analysis revealed that XynAS10-66 shared high similarity with a thermostable endo-beta-1,4-xylanase of Thermomonospora alba ULJB1 [41], which showed good activity at up to 95°C. Sequence alignment revealed that XynAS10-66 lacks a carbohydrate binding module (CBM) at the C terminal, which may have resulted in the lower temperature optimum of XynAS10-66. Future studies will be conducted to investigate this phenomenon. Moreover, other fragment sequences will be used to generate more full-length genes directly from the metagenomic DNA, and any genes generated will be characterized in subsequent studies.

Bottom Line: A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences.Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi.These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.

View Article: PubMed Central - PubMed

Affiliation: College of Biological Science and Technology, Fuzhou University, Fuzhou 350108, P.R. China; National Engineering Laboratory for High-efficiency Enzyme Expression, Fuzhou 350002, P. R. China.

ABSTRACT
Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.

Show MeSH
Related in: MedlinePlus