Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes

摘要

Mutant glycosidase enzymes are formed in which the normal nucleophilic amino acid within the active site has been changed to a nonnucleophilic amino acid. These enzymes cannot hydrolyze disaccharide products, but which can still form them. Using this enzyme, oligosaccharides are synthesized by preparing a mixture of anαglycosyl fluoride and a glycoside acceptor molecule; enzymatically coupling the &agr.glycosyl fluoride to the glycoside acceptor molecule to form a glycosyl glycoside product using the mutant glycosidase enzyme; and recovering the glycosyl glycoside product. Particular enzymes include a mutant form of Agrobacterium &bgr.Glucosidase in which the normal glutamic acid residue at position 358 is replaced with an alanine residue.