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

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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.

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Acknowledgements

This work was conducted under the research fund from the Agricultural Bioproducts Initiative Program (ABIP) Cellulosic Biofuels Network (CBioN) and Canadian Triticale Biorefinery Initiative (CTBI) of Agriculture and Agri-Food Canada and the genomics program of Genome Alberta. The authors would like to thank Krysty Munns for assistance with the editing and submission of this manuscript and Lyn Paterson for technical assistance.

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Authors and Affiliations

  1. Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada

    X. Gong, R. J. Gruniniger, R. J. Forster, R. M. Teather & T. A. McAllister

  2. Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China

    X. Gong

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  1. X. Gong
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  2. R. J. Gruniniger
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  3. R. J. Forster
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  4. R. M. Teather
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  5. T. A. McAllister
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Correspondence to T. A. McAllister.

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X. Gong and R. J. Gruniniger contributed equally to this work.

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Gong, X., Gruniniger, R.J., Forster, R.J. et al. Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library. Appl Microbiol Biotechnol 97, 2423–2431 (2013). https://doi.org/10.1007/s00253-012-4088-y

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  • DOI: https://doi.org/10.1007/s00253-012-4088-y

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