Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2001-11-19
2003-11-11
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S146000, C435S252330, C435S320100, C435S255400, C536S023200
Reexamination Certificate
active
06645746
ABSTRACT:
This application claims benefits of the International Application PCT/JP00/08321 filed Nov. 24, 2000, and the Japanese Patent Application 11345541 filed Dec. 3, 1999.
TECHNICAL FIELD
The present invention relates to a novel polypeptide, a gene encoding the polypeptide, an expression vector for expressing the polypeptide, a transformant obtained by transforming a host cell using the expression vector, and a method for producing a compound useful as a material for synthesis of medicaments and pesticides using the transformant. More particularly, the present invention relates to a polypeptide isolated from a microorganism having an enzyme activity to asymmetrically reduce tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, a polynucleotide encoding the polypeptide, an expression vector including the polynucleotide, and a transformant transformed by the expression vector.
The present invention also relates to a method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The method includes the steps of reacting the transformant or a processed product thereof with tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate, and collecting the produced tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.
Tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate is a compound useful as a material for synthesis of medicaments and pesticides, particularly HMG-CoA reductase inhibitors.
BACKGROUND ART
The only method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by asymmetrically reducing tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate, which has been reported, employs diethylmethoxyborane and sodium borohydride (U.S. Pat. No. 5,278,313). Problems with this technique are that a very low temperature reaction vessel achieving temperatures as low as −78° C. is required, that expensive materials need to be employed, and the like. There has been a demand for a practical reaction procedure.
DISCLOSURE OF THE INVENTION
The inventors of the present invention found a polypeptide derived from a microorganism which asymmetrically reduces tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to produce tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, envisioned a method which can efficiently produce tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, and eventually achieved the present invention.
An objective of the present invention is to provide a polypeptide capable of asymmetrically reducing tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to produce tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. Another objective of the present invention is to provide a method for efficiently producing the polypeptide using recombinant DNA technology. Still another objective of the present invention is to provide a transformant which can produce said polypeptide, and a polypeptide having a glucose dehydrogenase activity, simultaneously to a large extent. Even still another objective of the present invention is to provide a practical method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using the transformant.
The present invention relates to a polypeptide having an enzyme activity to asymmetrically reduce tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The polypeptide comprises the amino acid sequence represented by SEQ ID NO. 1 in the Sequence Listing, or the amino acid sequence having at least one amino acid substitution, insertion, deletion, or addition thereof.
Preferably, the peptide may be derived from a microorganism belonging to genus Candida. More preferably, the microorganism may be
Candida magnoliae
IFO 0705.
In one aspect, the present invention relates to a polynucleotide encoding the above-described polypeptide.
In one aspect, the present invention relates to a polynucleotide encoding a polypeptide having an enzyme activity to asymmetrically reduce tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The polynucleotide is hybridized with a nucleotide sequence represented by SEQ ID NO. 2 in the Sequence Listing under stringent conditions.
In one aspect, the present invention relates to an expression vector including the above-described polynucleotide. Preferably, the expression vector may be plasmid pNTCR.
In one embodiment, the above-described expression vector may further include a polynucleotide encoding a polypeptide having a glucose dehydrogenase activity.
Preferably, the polypeptide having a glucose dehydrogenase activity may be a glucose dehydrogenase derived from
Bacillus megaterium.
Preferably, the expression vector may be plasmid pNTCRG.
In one aspect, the present invention relates to a transformant obtained by transforming a host cell using the above-described expression vector.
Preferably, the host cell may be
Escherichia coli
. More preferably, the transformant may be
E. coli
HB101 (pNTCR) or
E. coli
HB101 (pNTCRG).
In one aspect, the present invention relates to a method for producing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The method comprises the steps of reacting the above-described transformant and/or a processed product thereof with tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate, and collecting produced tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.
Preferably, the reacting step is carried out in the presence of a coenzyme restoring system.
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patent: 00/36134 (2000-06-01), None
patent: 01/04336 (2001-01-01), None
Shimizu S. et al. Chiral alcohol synthesis with yeast carbonyl reductases, J. Mol. Catalysis B: Enzymatic, 1998, 5, 321-325.*
Shimizu S. et al. Chiral alcohol synthesis with microbial carbonyl reductases in a water-organic solvent two-phase system, Annals of the New York Academy of Sciences, 1998, 864, 87-95.*
Yasohara Y. Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase fromCandidia magnoliae, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, Biosci. Biotechnol. Biochem, 2000, 64, 1430-1436.1.*
M. Wada, et al.,“Purification and Characterization of NADPH-Dependent Carbonyl Reductase, Involved in Stereoselective Reduction of Ethyl 4-Choloro-3-oxobutanoate, from Candida magnoliae”, Biosci. Biotechnol. Biochem., 62(2):280-285 (1998).
Hasegawa Junzo
Kizaki Noriyuki
Yamada Yukio
Yasohara Yoshihiko
Achutamurthy Ponnathapu
Kaneka Corporation
Kenyon & Kenyon
Walicka Malgorzata
LandOfFree
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