Engineering lower inhibitor affinities in ß-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145.

ß-D-Xylosidase/α-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-ß-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (K (i) ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K(i)(D-glucose) and K(i)(D-xylose) twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k(cat)(D-xylobiose) and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K (i) values that are 1.5-2.7-fold (D-glucose) and 1.9-4.6-fold (D-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k(cat)(D-xylobiose) than that of the wild type. At 0.1 M D-xylobiose and 0.1 M D-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.