Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-10-22
2001-04-17
Lilling, Hebert J (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S071100, C435S191000, C435S280000
Reexamination Certificate
active
06218157
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel ketone reductase, a method for preparing the said enzyme, and a method for producing alcohols, especially (S)-4-halo-3-hydroxybutyric acid esters using the said enzyme.
BACKGROUND OF THE INVENTION
(S)-4-Halo-3-acetoacetic acid esters are important as HMG-CoA reductase inhibitors and intermediates for synthesizing various medicines and agricultural chemicals such as D-carnitine. The known methods for producing optically active 4-halo-3-hydroxybutyric acid esters include the asymmetric reduction methods using 3&agr;-hydroxysteroid dehydrogenase (Japanese Patent Laid-Open Publication (JP-A) No. Hei 1-1277494) or bakers' yeast (J. Am. Chem. Soc. 105, 5925-5926 (1983), and JP-A-Sho 61-146191).
Furthermore, the known methods for reducing 4-haloacetoacetic acid esters to produce 4-halo-3-hydroxybutyric acid esters include methods using enzymes such as
Saccharomyces cerevisiae
-derived D-enzyme-1 and D-enzyme-2 (J. Org. Chem. 56, 4778-4783 (1991)), L-enzyme-1 and L-enzyme-2 (Biosci. Biotech. Biochem. 58, 2236-2240 (1994)), aldehyde reductase derived from
Sporobolomyces salmonicolor
(Biochim. Biophys. Acta 1122, 57-62 (1992)), aldehyde reductase derived from Sporobolomyces sp. (Biosci. Biotech. Biochem. 57, 303-307 (1993)), aldehyde reductase derived from
Candida alcabins
(Biosci. Biotech. Biochem. 57, 303-307 (1993)), aldehyde reductase derived from
Trichosporon fermentans
(JP-A-Hei08-126487 (960521)), aldehyde reductase derived from
Hansenula mrakii
(JP-A-Hei08-126486 (960521)), ethyl ketopantothenate reductase derived from
Candida macedoniensis
(Arch. Biochem. Biophys. 294, 469-474 (1992)), and ethyl 4-chloroacetoacetate reductase derived from
Geotrichum candidum
(Enzyme Microb. Technol. 14, 731-738 (1992)). Among these methods, those using ethyl ketopantothenate reductase derived from
Candida macedoniensis,
D-enzyme-1 and D-enzyme-2 derived from
Saccharomyces cerevisiae,
and ethyl 4-chloroacetoacetate reductase derived from
Geotrichum candidum
produce the (S)-enatiomer of 4-halo-3-hydroxybutyric acid esters. However, these methods for producing (S)-4-halo-3-hydroxybutyric acid esters have the disadvantages of low optical purity and product yield.
The present inventors found that microorganisms including the genus Kluyveromyces reduce 4-haloacetoacetic acid esters to produce optically active 4-halo-3-hydroxybutyric acid esters and filed a patent application on the finding (JP-A-Hei 6-209782). However, the mechanism of the action of these microorganisms has not been clarified. Furthermore, this method enables producing highly optically pure (S)-4-halo-3-hydroxybutyric acid esters, but its product yield is unsatisfactory. When we tried to increase product yield, optical purity was lowered.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide a novel enzyme capable of reducing 4-haloacetoacetic acid esters to produce (S)-4-halo-3-hydroxybutyric acid esters with high optical purity. Another objective of this invention is to provide a method for producing (S)-4-halo-3-hydroxybutyric acid esters with high optical purity using the enzyme.
The present inventors observed the ability of various microorganisms to reduce 4-haloacetoacetic acid esters to produce optically active 4-halo-3-hydroxybutyric acid esters and ardently studied how to isolate an enzyme with such activity from the microorganisms. As a result, the inventors succeeded in isolating the desired enzyme from a microorganism belonging to the genus Kluyveromyces using various purification methods in combination. Furthermore, the present inventors succeeded in producing ethyl (S)-4-halo-3-hydroxybutyrate with high optical purity by reacting the enzyme thus isolated with ethyl 4-haloacetoacetate.
The present invention relates to a novel enzyme capable of reducing 4-haloacetoacetic acid esters to produce (S)-4-halo-3-hydroxybutyric acid esters, and a method for producing (S)-4-halo-3-hydroxybutyric acid esters with high optical purity using that enzyme. More specifically, it relates to:
(1) an enzyme having the following physicochemical properties:
(a) Function
The enzyme reduces 4-haloacetoacetic acid esters to produce (S)-4-halo-3-hydroxybutyric acid esters in the presence of &bgr;-nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor;
(b) Substrate specificity
The enzyme has high reducing activity on 4-chloro-3-acetoacetic acid esters, but does not act on acetoacetic acid esters. Furthermore, it has no dehydrogenase activity on (S)-4-halo-3-hydroxybutyric acid esters;
(c) Molecular weight
The enzyme's molecular weight is about 190,000 dalton when measured by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis;
(2) the enzyme as described in (1), wherein said enzyme is derived from a microorganism belonging to the genus Kluyveromyces,
(3) the enzyme as described in (2), wherein the microorganism belonging to the genus Kluyveromyces is selected from the group consisting of
Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces aestuarii,
and
Kluyveromyces yarrowii,
(4) the enzyme as described in (2), wherein the microorganism belonging to the genus Kluyveromyces is
Kluyveromyces lactis,
(5) a method for producing the enzyme of (1), wherein said method comprises culturing a microorganism containing said enzyme, and purifying it from the culture,
(6) the method as described in (5), wherein said microorganism belongs to the genus Kluyveromyces,
(7) the method as described in (5), wherein the purification comprises the steps of treatment with protamine sulfate, extraction from the protamine sulfate precipitates with a solution of a high salt concentration, precipitation with ammonium sulfate, hydrophobic chromatography, and gel filtration,
(8) a method for producing (S) -4-halo-3-hydroxybutyric acid esters, wherein said method comprises reacting 4-haloacetoacetic acid esters with the enzyme of (1) and recovering (S)-4-halo-3-hydroxybutyric acid esters, and
(9) the method as described in (8), wherein 4-haloacetoacetic acid esters are 4-chloroacetoacetic acid esters and (S)-4-halo-3-hydroxybutyric acid esters are (S)-4-chloro-3-hydroxybutyric acid esters.
REFERENCES:
patent: 5559030 (1996-09-01), Matsuyama et al.
patent: 6001618 (1999-12-01), Kimoto et al.
patent: 61-146191 (1986-07-01), None
Heidlas et al., “Purification and Characterization of two Oxidoreductases Involved in the Enantioselective Reduction of 3-oxo, 4-oxo and 5-oxo Esters in Baker's Yeast”, Eur. J. Biochem. 172:633-639, 1988.
Kataoka et al., “A Novel NADPH-Dependent Carbonyl Reductase of Candida Macedoniensis: Purification and Characterization”, Archives of Biochemistry and Biophysics 294:469-474, 1992.
Nakamura et al., “Stereochemical Control of Microbial Reduction. 17. A Method for Controlling the Enantioselectivity of Reductions with Bakers' Yeast”, J. Org. Chem. 56:4778-4783, 1991.
Patel et al., “Stereoselective Reduction of &bgr;-Keto Esters by Geotrichum Candidum”, Enzyme Microb. Technol. 14:731-738, 1992.
Shieh et al., “Stereochemical Control of Yeast Reductions. 5. Characterization of the OxidoreductasesInvolved in the Reduction of &bgr;-Keto Esters”, J. Am. Chem. Soc. 107:2993-2994, 1985.
Kimoto Norihiro
Yamamoto Hiroaki
Daicel Chemical Industries Ltd.
Fish & Richardson P.C.
Lilling Hebert J
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