Rapid screening of the aglycone specificity of glycosidases: applications to enzymatic synthesis of oligosaccharides

Abstract

Background: Retaining glycosidases can catalyse glycosidic bond formation through transglycosylation from a donor sugar to an acceptor bound in the aglycone site. The aglycone specificity of a glycosidase is not easily determined, thereby complicating the choice of the most appropriate glycosidase for use as a catalyst for transglycosylation. We have developed a strategy to rapidly screen the aglycone specificity of a glycosidase and thereby determine which enzymes are best suited to catalyse specific transglycosylation reactions.

Results: The reactivation, or turnover, of a glycosidase trapped as a fluoroglycosyl-enzyme species is accelerated in the presence of a compound that productively binds to the aglycone site. This methodology was used to rapidly screen six glycosidases with 44 potential acceptor sugars. Validation of the screening strategy was demonstrated by the identification of products formed from a transglycosylation reaction with positively screened acceptors for four of the enzymes studied.

Conclusions: The aglycone specificity of a glycosidase can be rapidly evaluated and requires only an appropriate fluorosugar inactivator, a substrate for assay of activity and a library of compounds for screening.