beta-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 (K (i) values of similar to 10(-2) M). Higher K (i)'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 1A degrees screen selects for activity (active/inactive screen) and the 2A degrees screen selects for a higher K (i(d-xylose)) and its subsequent use in screening similar to 5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K (i(d-xylose)) is threefold and K (i(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 from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite -1.