Cloning and enzymatic characterization of four thermostable fungal endo-1,4-β-xylanases

Appl Microbiol Biotechnol. 2014 Apr;98(8):3613-28. doi: 10.1007/s00253-013-5244-8. Epub 2013 Oct 2.

Abstract

Endo-1,4-β-xylanases (EC 3.2.1.8) hydrolyze the 1,4-β-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated GtXyn10A and GtXyn10B and two glycosyl hydrolase family 11 members, OpXyn11A and CcXyn11C, were functionally expressed and subjected to biochemical characterization. The K(M), V(max), and k(cat) values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 μmol/min/mg, and 109 to 344 s⁻¹, respectively, where OpXyn11A gave the highest and GtXyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between OpXyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of OpXyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basidiomycota / enzymology
  • Binding Sites
  • Cloning, Molecular
  • Endo-1,4-beta Xylanases / chemistry
  • Endo-1,4-beta Xylanases / genetics
  • Endo-1,4-beta Xylanases / metabolism*
  • Enzyme Stability / radiation effects
  • Kinetics
  • Models, Molecular
  • Ophiostoma / enzymology
  • Protein Conformation
  • Substrate Specificity
  • Temperature
  • Xylans / metabolism*

Substances

  • Xylans
  • Endo-1,4-beta Xylanases