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

Background: Retaining glycosidases can catalyse glycosidic bond formation t hrough transglycosylation from a donor sugar to an acceptor bound in the ag lycone site. The aglycone specificity of a glycosidase is not easily determ ined, thereby complicating the choice of the most appropriate glycosidase f or use as a catalyst for transglycosylation. We have developed a strategy t o rapidly screen the aglycone specificity of a glycosidase and thereby dete rmine which enzymes are best suited to catalyse specific transglycosylation reactions. Results: The reactivation, or turnover, of a glycosidase trapped as a fluor oglycosyl-enzyme species is accelerated in the presence of a compound that productively binds to the aglycone site. This methodology was used to rapid ly 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 acceptor s for four of the enzymes studied. Conclusions: The aglycone specificity of a glycosidase can be rapidly evalu ated and requires only an appropriate fluorosugar inactivator, a substrate for assay of activity and a library of compounds for screening. (C) 2001 El sevier Science Ltd. All rights reserved.