C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 9/24 (2006.01) C08B 37/00 (2006.01) C12N 9/26 (2006.01) C12P 19/04 (2006.01) C12P 19/14 (2006.01)
Patent
CA 2238966
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 .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.
L'invention concerne des enzymes du type glycosidases dans lesqueles l'aminoacide normal nucléophile dans le site actif a été changé en aminoacide non nucléophile. Ces enzymes ne peuvent pas hydrolyser les produits disaccharidiques, mais peuvent encore les former. Ces enzymes permettent de synthétiser des oligosaccharides à partir d'un mélange de fluorure d'.alpha.-glycosyle et d'un glycoside accepteur. On couple enzymatiquement, en utilisant l'enzyme glucosidase mutante, le fluorure d'.alpha.-glucosyle au glycoside accepteur pour former un glycosyl-glycoside et celui-ci est récupéré. Parmi les enzymes de l'invention, on peut citer une forme mutée de .beta.-glucosidase d'Agrobactérium dans laquelle l'aminoacide acide glutamique normal en position 358 est remplacé par l'aminoacide alanine
Mackenzie Lloyd
Wang Qingping
Withers Stephen G.
Smart & Biggar
The University Of British Columbia
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