Characterization of a thermostable, specific GH10 xylanase from Caldicellulosiruptor bescii with high catalytic activity

• A xylanase gene (xyn10B) was cloned and the enzyme was systematically characterized.
• CbXyn10B exhibited high xylanase activity and thermo-/pH stability.
• CbXyn10B displayed high substrate specificity and released mainly X2 from xylans.

Xylanase (EC 3.2.1.8) is one of the most important enzymes for the biodegradation of xylan. Since many industrial processes utilizing xylanase are operated at elevated temperatures, thermostable xylanases are highly desirable. In the present study, xyn10B gene from thermophilic bacterium Caldicellulosiruptor bescii that encodes a glycoside hydrolase (GH) family 10 xylanase was overexpressed in Escherichia coli and systematically characterized. CbXyn10B exhibited optimal activity at pH 7.2 and 70 °C. It had a half-life of about 7.7 h at 60 °C, and retained over 85% of maximal activity after incubation at pH 4.0–12.0. The activity of this xylanase was not affected by most divalent cations, but inhibited by Fe3+ and Zn2+. CbXyn10B exhibited high activity on beech wood xylan, oat spelt xylan, and birch wood xylan, with specific activities of about 450 U mg−1. Compared with other GH10 xylanases, CbXyn10B was highly specific for xylan and showed low catalytic efficiency toward sodium carboxymethyl cellulose and p-nitrophenyl-β-d-xylopyranoside. HPLC analysis of the products released from xylo-oligosaccharides and xylan revealed that xylobiose was the predominant hydrolytic product. The action mode of the enzyme was studied by product analysis, homology modeling and molecular docking to gain an insight into the structural basis for its substrate recognition mechanism.

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