Engineering lower inhibitor affinities in β-D-xylosidase

Zhanmin Fan, Ling Yuan, Douglas B. Jordan, Kurt Wagschal, Chamroeun Heng, Jay D. Braker

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


β-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 (Ki values of ∼10-2M). 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 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher Ki(D-xylose) and its subsequent use in screening ∼5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXAC3, Ki(D-xylose) is threefold and Ki(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 Å from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite -1.

Original languageEnglish
Pages (from-to)1099-1113
Number of pages15
JournalApplied Microbiology and Biotechnology
Issue number4
StatePublished - Apr 2010

Bibliographical note

Funding Information:
Acknowledgments This work was supported by US Department of Agriculture CRIS 5325-41000-046-00 (K.W. and C.H), CRIS 3620-41000-118-00D (D.B.J. and J.D.B.), and CSREES 2006-35504-17413 (to L.Y.). The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.


  • Bioenergy
  • Glycoside hydrolase
  • Protein engineering
  • Selection
  • β-D-Xylosidase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology


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