|FAN, ZHANMIN - University Of Kentucky|
|YUAN, LING - University Of Kentucky|
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2009
Publication Date: 1/5/2010
Citation: Fan, Z., Yuan, L., Jordan, D.B., Wagschal, K.C., Heng, C., Braker, J.D. 2010. Engineering lower inhibitor affinities in beta-D-xylosidase. Applied Microbiology and Biotechnology. 86(4):1099-1113.
Interpretive Summary: The United States produces nearly 3 billion gallons containing 2.28X1014 BTU of ethanol annually from grain. This work is part of a larger effort to expand the biomass sources used to make ethanol to include crop residues. Hemicellulose is a major chemical constituent of crop residues, and this work will help define new options for biorefining processes to reduce the chemical and energy costs associated with ethanol production, such that the use of crop residues is economically feasible. The project strategy starts with the identification of enzymes that are able to degrade hemicellulose to fermentable sugars, which can then be readily converted to ethanol or other chemical feedstocks. To do so enzymatically requires a suite of different enzymes, a critical one of which is beta-xylosidase. We describe here protein engineering using directed evolution of the xylosidase SXA for improved resistance to end-product monosaccharide inhibition.
Technical Abstract: Beta- 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 ~10-5 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 primary screen selects for activity (active/inactive screen) and the secondary screen selects for a higher Ki(D-xylose), and its subsequent use in screening ~5900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, Ki(D-xylose) is 3-fold and Ki(D-glucose) is 2-fold 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 the changed affinity stems from changed affinity for D-xylose in subsite +1 and not in subsite -1 of the two-subsite active site. W145 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.