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

Singh, Sanjay; Heng, Chamroeun; Braker, Jay; Chan, Victor; Lee, Charles; Jordan, Douglas; Yuan, Ling; Wagschal, Kurt

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

XYLOSIDASES; GLYCOSIDASES; THERMAL stability; MUTAGENESIS; GENETIC mutation; XYLOSE

Journal of Industrial Microbiology & Biotechnology, 2014, Vol 41, Issue 3, p489

1367-5435

Academic Journal

10.1007/s10295-013-1377-0