Novel carbonyl reductase, gene thereof and method of using...

Details Novel carbonyl reductase, gene thereof and method of using... Novel carbonyl reductase, gene thereof and method of using...

C - Chemistry, Metallurgy

12

N

C12N 15/53 (2006.01) C12N 1/21 (2006.01) C12N 9/02 (2006.01) C12N 9/04 (2006.01) C12P 17/10 (2006.01) C12P 41/00 (2006.01)

Patent

CA 2384625

Novel polypeptides capable of forming (S)-N-benzyl-3-pyrrolidinol, DNA encoding the same and a method of using the same. A polypeptide having the following physicochemical properties (1) to (5): (1) function: asymmetrically reducing N-benzyl-3-pyrrolidinone by using NADPH as a coenzyme to form (S)-N- benzyl-3-pyrodinol; (2) optimum functional pH: 4.5 to 5.5; (3) optimum functional temperature: 40 to 45~C; (4) molecular weight: about 29,000 in gel filtration analysis, about 35,000 in SDS polyacrylamide electrophoresis; and (5) inhibitor: being inhibited by divalent copper ion. A polypeptide having the amino acid sequence represented by SEQ ID NO:1, or a polypeptide having an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by the substitution, insertion, deletion and/or addition of one or more amino acids and having an enzymatic activity of asymmetrically reducing N- benzyl-3-pyrrolidinone to form (S)-N-benzyl-3-pyrrolidinol.

L'invention concerne de nouveaux polypeptides capables de former du (S)-N-benzyl-3-pyrrolidinol, l'ADN codant ce dernier et un procédé d'utilisation desdits polypeptides. Ce polypeptide présente les propriétés physico-chimiques suivantes : (1) fonction : réduction asymétrique de la N-benzyl-3-pyrrolidinone au moyen de NADPH comme coenzyme pour former le (S)-N-benzyl-3-pyrrolidinol ; (2) pH fonctionnel optimal : 4,5 à 5,5 ; (3) température fonctionnelle optimale : 40 à 45 ·C ; (4) poids moléculaire : environ 29 000 dans les analyses de filtration sur gel, environ 35 000 dans l'électrophorèse sur gel de polyacrylamide SDS ; et (5) inhibiteur : inhibition par ion cuivre divalent. L'invention concerne également un polypeptide présentant la séquence d'acide aminé représentée par le numéro d'identification de séquence 1, ou un polypeptide présentant une séquence d'acide aminé dérivée de cette dernière, par substitution, insertion, délétion et/ou addition d'un ou plusieurs acides aminés et présentant une activité enzymatique qui permet de réduire de façon asymétrique la N-benzyl-3-pyrrolidinone pour former le (S)-N-benzyl-3-pyrrolidinol.

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