Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis
- First Online:
- Cite this article as:
- Singh, S.K., Heng, C., Braker, J.D. et al. J Ind Microbiol Biotechnol (2014) 41: 489. doi:10.1007/s10295-013-1377-0
- 393 Downloads
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 Kt0.5 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 Kt0.5 by ~3 °C; none of the individual amino acid changes measurably affect Kt0.5. Saturation mutagenesis of L186 identified the variant L186K as having the most improved Kt0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in Kt0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While kcat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, Km, Kss (substrate inhibition), and Ki (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.