Monday, April 19, 2010
3-50

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

Douglas B. Jordan1, Kurt Wagschal2, Zhanmin Fan3, Ling Yuan3, Jay Braker1, and Chamroeun Heng2. (1) Bioenergy Research, USDA-ARS, 1815 N University Street, Peoria, IL 61604, (2) Bioproduct Chemistry and Engineering, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, (3) Kentucky Tobacco Research and Development Center, Department of Plant and Soil Sciences, University of Kentucky, Cooper & University Drives, Lexington, KY 40546

β‑Xylosidase from Selenomonas ruminantium, SXA, is the most active catalyst known for promoting hydrolysis of xylooligosaccharides to D‑xylose monosaccharides.  Its activity is inhibited by D‑xylose and D‑glucose (Ki values of ~10-2 M), and higher Ki’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production and other purposes.  We developed a two-tier high-throughput screen for screening an SXA enzyme library prepared using error-prone PCR.  The primary screen selects for activity (active/inactive screen) and the secondary screen selects for a higher Ki(D‑xylose).  The screen led to the discovery of a SXA variant, W145G, in which Ki(D-xylose) is 3-fold and Ki(D‑glucose) is 2-fold that of wild-type SXA.  Site-directed mutagenesis was used to replace W145 with the remaining 18 natural-occurring amino acids.  Kinetic parameters, Ki values for D‑xylose and D‑glucose, and stabilities to pH and temperature of the single-site variants will be reported.

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