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

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

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.

Original languageEnglish
Pages (from-to)489-498
Number of pages10
JournalJournal of Industrial Microbiology and Biotechnology
Volume41
Issue number3
DOIs
StatePublished - Mar 2014

Bibliographical note

Funding Information:
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.

Keywords

  • Directed evolution
  • Gene shuffling
  • Glycosyl hydrolase
  • Protein engineering
  • Thermal stability

ASJC Scopus subject areas

  • General Medicine

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