title: Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library.
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 Mn(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.
authors.affiliation: Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada.
authors.forename: X
authors.initials: X
authors.lastname: Gong
authors.forename: R J
authors.initials: RJ
authors.lastname: Gruniniger
authors.forename: R J
authors.initials: RJ
authors.lastname: Forster
authors.forename: R M
authors.initials: RM
authors.lastname: Teather
authors.forename: T A
authors.initials: TA
authors.lastname: McAllister
mesh_terms: D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D017124:Conserved Sequence; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012417:Rumen / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D043364:Endo-1,4-beta Xylanases