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A glycosynthase is a mutant glycoside hydrolase that can be used to synthesize the glycosidic bond in high yield.

Glycosynthases are typically formed from retaining glycoside hydrolases (or glycosidases) by mutating the enzymic nucleophile from a carboxylate group to another, usually non-nucleophilic, residue such as glycine or alanine. Such mutant enzymes are severely diabled catalysts and cannot catalyze the cleavage of normal substrates. However, when presented with an activated sugar of the 'wrong' anomeric configuration, such as a glycosyl fluoride, glycosynthases can catalyze the condensation of sugar residues, synthesizing a glycoside. The newly formed glycoside, being a poor substrate for the mutant enzyme, can accumulate to essentially quantitative yields.