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.
Bibliographical noteFunding 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.