数字农科院2.0

Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library

文献类型： 外文期刊

作者： Gong, X.;Gruniniger, R. J.;Forster, R. J.;Teather, R. M.;McAllister, T. A.;Gong, X.

作者机构：

关键词： Xylanase;Cow ruminal microorganisms;BAC library;Glycosyl hydrolase (GH) 10;Biobleaching

期刊名称： APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

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收录情况： SCI

摘要： A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four openreading frames (ORFs). The ORF, xyn10N18, encodes an endo-β-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/?8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0–12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of>40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cationsMn~(2+) and Co~(2+) and is dramatically reduced by Hg~(2+) and Cu~(2+). Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry.

分类号： Q939.9

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