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

Content Provider	Springer Nature Link
Author	Gong, X. Gruniniger, R. J. Forster, R. J. Teather, R. M. McAllister, T. A.
Copyright Year	2012
Abstract	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 open-reading 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/ß8-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 cations Mn2+ and Co2+ and is dramatically reduced by Hg2+ and Cu2+. 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.
Starting Page	2423
Ending Page	2431
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	6
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-04-26
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Xylanase Cow ruminal microorganisms BAC library Glycosyl hydrolase (GH) 10 Biobleaching Biotechnology Microbiology Microbial Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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