A Universal Screening Assay for Glycosynthases: Directed Evolution of Glycosynthase XynB2(E335G) Suggests a General Path to Enhance Activity

Alon Ben-David, Gil Shoham, Yuval Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

Abstract

Glycosynthases are catalytic mutants of mainly retaining glycoside hydrolases that catalyze the synthesis of oligosaccharides from their corresponding glycosyl-fluoride donors and suitable acceptors. Here we describe the development of a general, high-throughput screening procedure for glycosynthase activity, which is based on the release of hydrofluoric acid, a by-product of all glycosynthase reactions. This assay is sensitive, does not require the synthesis of special chromophoric or modified substrates, and, most importantly, is applicable for all glycosynthases. We used this screening procedure on error-prone PCR libraries to isolate improved glycosynthase variants of XynB2(E335G) glycosynthase, a family 52 β-xylosidase from Geobacillus stearothermophilus. The improved variants exhibited higher KM values toward the acceptor and the donor, suggesting that enzyme-product release is rate determining for kcat.

Original languageAmerican English
Pages (from-to)546-551
Number of pages6
JournalChemistry and Biology
Volume15
Issue number6
DOIs
StatePublished - 23 Jun 2008

Bibliographical note

Funding Information:
This study was supported by grants from the German-Israeli Foundation for Scientific Research and Development (no. 743-119), and from the Israel Science Foundation (no. 676-00 to G.S. and Y.S.; no. 1006-05 to Y.S.). Additional support was provided by the Otto Meyerhof Center for Biotechnology, Technion, established by the Minerva Foundation (Munich, Germany). Y.S. holds the Erwin and Rosl Pollak Chair in Biotechnology.

Keywords

  • CHEMBIO

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