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 language | English |
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Pages (from-to) | 489-498 |
Number of pages | 10 |
Journal | Journal of Industrial Microbiology and Biotechnology |
Volume | 41 |
Issue number | 3 |
DOIs | |
State | Published - 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.
Funding
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.
Funders | Funder number |
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U.S. Department of Agriculture | CRIS 5325-41000-049-00, CRIS 3620-41000-118-00D |
Keywords
- Directed evolution
- Gene shuffling
- Glycosyl hydrolase
- Protein engineering
- Thermal stability
ASJC Scopus subject areas
- General Medicine