| 摘要 |
A method for modifying an enzyme whereby the coenzyme-dependency of an oxidoreductase is changed by controlling the size of binding energy of the coenzyme through substitution, insertion or deletion or 1 or more amino acid residues in a pre-selected part of the oxidoreductase, or their combination. Independent claims are also included for the following: (1) oxidoreductase variants thus modified; (2) carbonyl reductase variants in which their coenzyme-dependency is changed by the enzyme-modification method; (3) carbonyl reductase variants in which the parents have been modified by substitution, insertion or deletion of some amino acid residues or their combination, and having physiochemical properties of (a) using the reduced form beta -nicotinamide adenine dinucleotide as coenzyme for acting on a 4-chloroacetoacetic acid ester to give an (S)-4-chloro-3-hydroxybutyric acid ester; (b) exhibiting potent activity on a 4-chloroacetoacetic acid ester and practically not on acetoacetic acid ester, and without substantial dehydrogenase activity on a 4-halo-3-hydroxybutyric acid ester; and (c) exhibiting potent activity when applying the reduced form beta -nicotinamide adenine dinucleotide but without substantial activity when using the reduced form beta -nicotinamide adenine dinucleotide phosphate as coenzyme; (4) DNAs encoding the enzyme variants; (5) plasmids containing the DNAs; (6) transformant cells which are transformed by the plasmids; (7) producing the carbonyl reductase variant by culturing and growing the transformant cells; (8) a process for producing an (S)-4-chloro-3-hydroxybutyric acid ester of formula (I) by reaction a 4-haloacetoacetic acid ester with the enzyme variant, or the culture of a microorganism capable of producing such enzyme variant or its processed material; and (I) and (II) (9) producing an optically-active alcohol by reacting a carbonyl compound with any of the enzyme variants and an enzyme capable of regenerating the enzyme variant-dependent coenzyme and/or its variant, followed by collecting the product; or by reacting a carbonyl compound with transformant cells which are transformed with a plasmid containing a DNA encoding any of the enzyme variant and a DNA encoding an enzyme capable of regenerating the enzyme variant-dependent coenzyme, and collecting the produced optically-active alcohol. [Image] R1 : halo R2 : H R3 : (un)substituted alkyl or aryl. |
