title: Engineering lower inhibitor affinities in β-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145.
abstract: β-D-Xylosidase/α-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-β-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.
authors.affiliation: National Center for Agricultural Utilization Research, USDA Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA. douglas.jordan@ars.usda.gov
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
authors.forename: Kurt
authors.initials: K
authors.lastname: Wagschal
authors.forename: Zhanmin
authors.initials: Z
authors.lastname: Fan
authors.forename: Ling
authors.initials: L
authors.lastname: Yuan
authors.forename: Jay D
authors.initials: JD
authors.lastname: Braker
authors.forename: Chamroeun
authors.initials: C
authors.lastname: Heng
mesh_terms: D055162:Biocatalysis; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020357:Selenomonas / Q000201:enzymology*; D014364:Tryptophan / Q000235:genetics; D014994:Xylose / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D014364:Tryptophan; D014994:Xylose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase; D005947:Glucose