Engineering lower inhibitor affinities in β- d-xylosidase.

Zhanmin Fan; Ling Yuan; Jordan, Douglas B.; Wagschal, Kurt; Chamroeun Heng; Braker, Jay D.

β- d-Xylosidase catalyzes hydrolysis of xylooligosaccharides to d-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by d-xylose and d-glucose ( Ki values of ∼10−2 M). Higher Ki’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher Ki( d-xylose) and its subsequent use in screening ∼5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, Ki( d-xylose) is threefold and Ki( d-glucose) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite −1 or subsite 1 indicate that the changed affinity stems from changed affinity for d-xylose in subsite 1 and not in subsite −1 of the two-subsite active site. Trp145 is 6 Å from the active site, and its side chain contacts three active-site residues, two in subsite 1 and one in subsite −1.

XYLOSIDE; HYDROLYSIS; GLUCOSE; LIGNOCELLULOSE; ETHANOL as fuel; AMINO acids; PROTEIN engineering; GLYCOSIDASES; BIOMASS energy; MONOSACCHARIDES; ENZYMES

Applied Microbiology & Biotechnology, 2010, Vol 86, Issue 4, p1099

0175-7598

Academic Journal

10.1007/s00253-009-2335-7