Carbonyl reductase, gene thereof and use of the same
View Patent ↗The present invention provides a novel polypeptide forming (R)-2-chloro-1-(3′-chlorophenyl)ethanol, a polynucleotide coding for said polypeptide, and use of the same. The present invention relates to a polypeptide having the following physical and chemical properties (1) to (4): (1) activity: acting on 2-chloro-1-(3′-chlorophenyl)ethanone with NADPH or NADH as a coenzyme, to form (R)-2-chloro-1-(31-chlorophenyl)ethanol; (2) optimum pH for activity: 5.0 to 6.0; (3) optimum temperature for activity: 40° C. to 50° C.; (4) molecular weight: about 40,000 as determined by gel filtration analysis, about 30,000 as determined by SDS polyacrylamide gel electrophoresis analysis. The present invention also relates to a polypeptide comprising the amino acid sequence shown under SEQ ID NO:1 in the sequence listing, a polynucleotide coding for said polypeptide, and a transformant producing said polypeptide at high levels.
1. An isolated polynucleotide encoding a polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:1.
2. An isolated polynucleotide wherein:
the polynucleotide comprises SEQ ID NO:2, or
the polynucleotide is capable of hybridizing under stringent conditions with the nucleotide sequence complementary to the full length of SEQ ID NO:2 and encoding a polypeptide having activity in asymmetrically reducing 2-chloro-1-(3′-chlorophenyl)ethanone represented by the formula (1):
to form (R)-2-chloro-1-(3′-chlorophenyl)ethanol represented by the formula (2):
wherein said stringent conditions involve washing a filter with 0.1 to 2×SSC solution at a temperature of 65° C.
3. An expression vector comprising the polynucleotide according to claim 2 .
4. The expression vector according to claim 3 , wherein the expression vector is a plasmid pNTRG.
5. The expression vector according to claim 3 , further comprising a polynucleotide encoding a polypeptide having glucose dehydrogenase activity.
6. The expression vector according to claim 5 , wherein the polypeptide having glucose dehydrogenase activity is a Bacillus megaterium -derived glucose dehydrogenase.
7. The expression vector according to claim 6 , wherein the expression vector is a plasmid pNTRGG1.
8. A transformant, resulting from transforming an isolated host cell with the expression vector according to claim 3 .
9. The transformant according to claim 8 , wherein the host cell is Escherichia coli ( E. coli ).
10. The transformant according to claim 9 , wherein the transformant is E. coli HB101(pNTRG) (FERM BP-7857).
11. The transformant according to claim 9 , wherein the transformant is E. coli HB101(pNTRGG1) (FERM BP-7858).
12. The isolated polynucleotide according to claim 2 , wherein the polypeptide is derived from a microorganism belonging to the genus Rhodotorula.
13. The isolated polynucleotide according to claim 12 , wherein the microorganism belonging to the genus Rhodotorula is Rhodotorula glutinis var. dairenensis IFO 0415.
14. A method of producing an optically active 1-phenylethanol derivative represented by the formula (4), comprising reacting the culture of the transformant according to claim 8 , or a processed product thereof with a carbonyl group-containing compound represented by formula (3):
wherein,
R 1 and R 2 may be the same or different and each represents a hydrogen or halogen atom or an alkoxy or nitro group,
R 3 represents a hydrogen or halogen atom, a hydroxyl group or an alkyl group, which may optionally be substituted, the formula (4) being:
wherein, R 1 , R 2 and R 3 are as defined above.
15. The method according to claim 14 , wherein the carbonyl group-containing compound is 2-chloro-1-(3'-chlorophenyl)ethanone represented by the formula (1):
and
the optically active alcohol is (R)-2-chloro-1-(3'-chlorophenyl)ethanol represented by the formula (2):
16. The method according to claim 14 , wherein the carbonyl group-containing compound is 1-(2'-fluorophenyl)ethanone represented by the formula (5):
and
the optically active alcohol is (S)-1-(2'-fluorophenyl)ethanol represented by the formula (6):
17. A method for producing optically active 3-hydroxyester derivative represented by formula (8), comprising reacting the culture of the transformant according to claim 8 , or a processed product thereof with a carbonyl group-containing compound represented by formula (7):
wherein,
R 4 represents a hydrogen or halogen atom, an azido or benzyloxy group or an alkyl group, which may optionally be substituted, and
R 5 represents an alkyl or phenyl group, the general formula (8) being:
wherein, R 4 and R 5 are as defined above.
18. the method according to claim 17 , wherein the carbonyl group-containing compound is ethyl 4-chloroacetoacetate represented by the formula (9):
and
the optically active alcohol is ethyl (R)-4-chloro-3-hydroxybutyrate represented by the formula (10):