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

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 K t 0.5 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 K t 0.5 by ~3 °C; none of the individual amino acid changes measurably affect K t 0.5 . Saturation mutagenesis of L186 identified the variant L186K as having the most improved K t 0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K t 0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k cat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K m, K ss (substrate inhibition), and K i (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Keywords

Glycosyl hydrolase Directed evolution Gene shuffling Thermal stability Protein engineering 

Notes

Acknowledgments

This work was supported by funding from the United States Department of Agriculture, CRIS 5325-41000-049-00 (C.H., V.J.C., C.C.L., K.W.) and CRIS 3620-41000-118-00D (D.B.J. and J.D.B.). The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. The USDA is an equal opportunity provider and employer.

Supplementary material

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Supplementary Table 1 (DOCX 10 kb)
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Supplementary Table 2 (DOCX 24 kb)
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Supplementary Fig. 1 (DOCX 10 kb)

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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

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