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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Publications at this Location » Publication #250485
Title: Engineering lower inhibitor affinities in beta-D-xylosidase by site-directed mutagenesis of Trp 145

Author
item Jordan, Douglas
item Wagschal, Kurt
item FAN, ZHANMIN - University Of Kentucky
item YUAN, LING - University Of Kentucky
item Braker, Jay
item Heng, Chamroeun

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/7/2009
Publication Date: 4/19/2010
Citation: Jordan, D.B., Wagschal, K.C., Fan, Z., Yuan, L., Braker, J.D., Heng, C. 2010. Engineering lower inhibitor affinities in beta-D-xylosidase by site-directed mutagenesis of Trp 145. Meeting Abstract. 84.

Interpretive Summary:

Technical Abstract: Beta- D-xylosidase catalyzes hydrolysis of xylooligosaccharides to D-xylose monosaccharides. beta-Xylosidase from Selenomonas ruminantium, SXA, 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 developed a two-tier high-throughput screen, where the primary screen selects for activity (active/inactive screen) and the secondary screen selects for a higher Ki(D xylose), for screening an SXA enzyme library prepared using error-prone PCR. 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 amino acids and kinetic parameters and Ki values of the single-site variants will be reported.