ARS | Publication request: Engineering lower inhibitor affinities in beta-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 31, 2011
Publication Date: June 1, 2011
Citation: Jordan, D.B., Wagschal, K.C., Zhanmin, F., Yuan, L., Braker, J.D., Heng, C. 2011. Engineering lower inhibitor affinities in beta-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145. Journal of Industrial Microbiology and Biotechnology. 38:1821-1835.

Interpretive Summary: Agricultural biomass such as crop residues, grain processing byproducts, dedicated energy crops (e.g., switchgrass), etc., represent abundant, renewable feedstocks for production of ethanol and other valuable products if practical conversion technologies can be developed. These materials are rich in complex carbohydrates that must first be broken down to simple sugars that can be fermented by microorganisms to ethanol and other products. A critical step in the development of new conversion processes is the discovery and development of cost efficient enzymes to convert these complex materials to simple sugars. We have discovered an enzyme involved in the final step in the hydrolysis of xylan, the second most abundant carbohydrate in plants. This enzyme produces the simple sugar, D-xylose, more efficiently than counterpart enzymes described by other workers. The enzyme also catalyzes the release of L-arabinose from arabinosides. This work reveals site-directed mutagenesis experiments to discover enzyme variants that have lower affinities (less inhibition) for D-xylose and D-glucose so that the enzyme can operate more efficiently. Our results will help the development of new bioconversion strategies to produce fuel ethanol economically.

Technical Abstract: Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (Ki~10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses Ki**D-glucose and Ki**D-xylose 2-fold and 3-fold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower kcat**D-xylobiose and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express Ki values that are 1.5- to 2.7-fold (D-glucose) and 1.9- to 4.6-fold (D-xylose) of those for the wild-type enzyme. W145F, W145L, and W145Y express good stability and respectively 11%, 6%, and 1% higher kcat**D-xylobiose than that of 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.