Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis.
J Ind Microbiol Biotechnol
; 41(3): 489-98, 2014 Mar.
Article
en En
| MEDLINE
| ID: mdl-24292973
ABSTRACT
Directed evolution of ß-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K(t)°·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)°·5 by ~3 °C; none of the individual amino acid changes measurably affect K(t)°·5. Saturation mutagenesis of L186 identified the variant L186K as having the most improved K(t)°·5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K(t)°·5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k cat of xylobiose and 4-nitrophenyl-ß-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.
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Banco de datos:
MEDLINE
Asunto principal:
Bacillus
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Xilosidasas
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Evolución Molecular Dirigida
Idioma:
En
Año:
2014
Tipo del documento:
Article