Engineering lower inhibitor affinities in beta-D-xylosidase

Applied Microbiology and Biotechnology
Zhanmin FanJay D Braker

Abstract

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 approximately 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 1 degrees screen selects for activity (active/inactive screen) and the 2 degrees screen selects for a higher K (i(D-xylose)) and its subsequent use in screening approximately 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 A from the active...Continue Reading

References

Nov 1, 1989·Analytical Biochemistry·S C Gill, P H von Hippel
Feb 26, 2000·Proceedings of the National Academy of Sciences of the United States of America·P S DaughertyG Georgiou
Feb 10, 2004·Protein Science : a Publication of the Protein Society·Steven C RothmanJack F Kirsch
Jul 21, 2004·Journal of Computational Chemistry·Eric F PettersenThomas E Ferrin
Jan 13, 2005·Proceedings of the National Academy of Sciences of the United States of America·Jesse D BloomFrances H Arnold
Apr 7, 2005·Current Opinion in Chemical Biology·Amir AharoniDan S Tawfik
Jun 9, 2005·Journal of Molecular Biology·D Allan DrummondFrances H Arnold
Sep 9, 2005·Microbiology and Molecular Biology Reviews : MMBR·Ling YuanRobert Keenan
Jul 13, 2007·Applied Biochemistry and Biotechnology·Douglas B JordanMichael A Cotta
Mar 15, 2008·Applied Microbiology and Biotechnology·Moon-Soo Kim, Xin Gen Lei
May 15, 2008·Applied Biochemistry and Biotechnology·Douglas B JordanMichael A Cotta
Sep 26, 2008·Applied Biochemistry and Biotechnology·Kurt WagschalDominic W S Wong
Nov 1, 2008·Biochimica Et Biophysica Acta·Douglas B JordanJay D Braker

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Citations

Oct 12, 2012·Applied Microbiology and Biotechnology·Douglas B JordanJay D Braker
Jan 31, 2012·Journal of Microbiological Methods·Kurt Wagschal, Charles C Lee
Dec 3, 2013·Journal of Industrial Microbiology & Biotechnology·Sanjay K SinghKurt Wagschal
Apr 30, 2011·Journal of Industrial Microbiology & Biotechnology·Douglas B JordanChamroeun Heng
Jan 1, 2013·Enzyme and Microbial Technology·Charles C LeeDouglas B Jordan
Nov 9, 2019·International Journal of Molecular Sciences·Ali RohmanNi Nyoman Tri Puspaningsih

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