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... More
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.
