Journal of Industrial Microbiology & Biotechnology

, Volume 41, Issue 3, pp 489–498

Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis

  • Sanjay K. Singh
  • Chamroeun Heng
  • Jay D. Braker
  • Victor J. Chan
  • Charles C. Lee
  • Douglas B. Jordan
  • Ling Yuan
  • Kurt Wagschal
Bioenergy/Biofuels/Biochemicals

DOI: 10.1007/s10295-013-1377-0

Cite this article as:
Singh, S.K., Heng, C., Braker, J.D. et al. J Ind Microbiol Biotechnol (2014) 41: 489. doi:10.1007/s10295-013-1377-0

Abstract

Directed evolution of β-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter Kt0.5 by −1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved Kt0.5 by ~3 °C; none of the individual amino acid changes measurably affect Kt0.5. Saturation mutagenesis of L186 identified the variant L186K as having the most improved Kt0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in Kt0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While kcat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, Km, Kss (substrate inhibition), and Ki (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Keywords

Glycosyl hydrolaseDirected evolutionGene shufflingThermal stabilityProtein engineering

Supplementary material

10295_2013_1377_MOESM1_ESM.docx (11 kb)
Supplementary Table 1 (DOCX 10 kb)
10295_2013_1377_MOESM2_ESM.docx (25 kb)
Supplementary Table 2 (DOCX 24 kb)
10295_2013_1377_MOESM3_ESM.docx (11 kb)
Supplementary Fig. 1 (DOCX 10 kb)

Copyright information

© Springer-Verlag (outside the USA) 2013

Authors and Affiliations

  • Sanjay K. Singh
    • 2
  • Chamroeun Heng
    • 1
  • Jay D. Braker
    • 3
  • Victor J. Chan
    • 1
  • Charles C. Lee
    • 1
  • Douglas B. Jordan
    • 3
  • Ling Yuan
    • 2
  • Kurt Wagschal
    • 1
  1. 1.USDA Agricultural Research ServiceWestern Regional Research CenterAlbanyUSA
  2. 2.Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development CenterUniversity of KentuckyLexingtonUSA
  3. 3.USDA Agricultural Research ServiceNational Center for Agricultural Utilization ResearchPeoriaUSA