Abstract: Mutant glycosidase enzymes are formed in which the normal nucleophilic amino acid within the active site has been changed to a non-nucleophilic 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 an &agr;-glycosyl 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.

Abstract: Glycosylated acceptors are prepared using glycosyl transferase and activated glycosyl derivatives as donor sugars without the use of sugar nucleotides as donor sugars. A reaction mixture composition containing an activated glycoside derivative such as glycosyl fluoride or glycosyl mesylate, an acceptor substrate such as lactose or other oligosaccharide, a glycosyl transferase and a catalytic amount of a nucleotide phosphate or nucleotide phosphate analog is reacted to produce the glycosylated acceptor. The acceptor substrate may also be a monosaccharide, a fluorescent-labeled saccharide or a saccharide derivative such as an aminoglycoside antibiotic. The glycosyl transferase may be immobilized by removing its membrane-binding domain and attaching in its place a cellulose-binding domain. In another embodiment, a glycosylated acceptor is formed by making a nucleotide phosphate glycoside in situ in a steady state concentration.

Abstract: Oligosaccharides are prepared using glycosyl transferase and activated glycosyl derivatives as donor sugars without the use of sugar nucleotides as donor sugars. A reaction mixture composition containing an activated glycoside derivative such as glycosyl fluoride or glycosyl mesylate, an acceptor substrate such as lactose or other oligosaccharide, a glycosyl transferase and a catalytic amount of a nucleotide phosphate or nucleotide phosphate analog is reacted to produce a glycosylated acceptor. In addition to an oligosaccharide, the acceptor substrate may be a monosaccharide, a fluorescent-labeled saccharide or a saccharide derivative such as an aminoglycoside antibiotic. The glycosyl transferase may be immobilized by removing its membrane-binding domain and attaching in its place a cellulose-binding domain. In a preferred embodiment, galactosyl transferase is the glycosyl transferase, .alpha.-D-galactosyl fluoride is the activated glycoside derivative and lactose is the acceptor substrate.

Abstract: Mutant glycosidase enzymes are formed in which the normal nucleophilic amino acid within the active site has been changed to a non-nucleophilic amino acid. These enzymes cannot hydrolyze disaccharide products, but can still form them. Using this enzyme, oligosaccharides are synthesized by preparing a mixture of an .alpha.-glycosyl fluoride and a glycoside acceptor molecule; enzymatically coupling the .alpha.-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 .beta.-Glucosidase in which the normal glutamic acid residue at position 358 is replaced with an alanine residue.