Carbonyl reductase, gene thereof and method of using the same
View Patent ↗The present invention relates to a polypeptide having an activity to asymmetrically reduce (3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanone to produce (2R,3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol isolated from a microorganism belonging to the genus Ogataea , a DNA encoding the polypeptide and a transformant that produces the polypeptide. The present invention moreover relates to a method of producing (2R,3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol utilizing the polypeptide or the transformant. Using the polypeptide or transformant of the present invention, optically active alcohols such as (2R,3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol and the like can be produced efficiently.
1. An isolated DNA comprising the base sequence of SEQ ID NO: 2.
2. An isolated DNA encoding a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, and
(b) a polypeptide having the amino acid sequence of SEQ ID NO: 4 except for at least one modification in SEQ ID NO: 4 selected from the group consisting of:
(1) the alanine at position 42 of SEQ ID NO: 4 is substituted by glycine,
(2) the glutamic acid at position 43 of SEQ ID NO: 4 is substituted by alanine,
(3) the lysine at position 46 of SEQ ID NO: 4 is substituted by glutamic acid, and
(4) the asparagine at position 49 of SEQ ID NO: 4 is substituted by lysine.
3. A vector comprising the DNA of claim 1 .
4. The vector of claim 3 , further comprising a DNA encoding a polypeptide having a glucose dehydrogenase activity.
5. The vector of claim 4 , wherein the polypeptide having a glucose dehydrogenase activity is a glucose dehydrogenase derived from Bacillus megaterium.
6. An isolated host cell transformed with the vector of claim 3 .
7. The isolated host cell of claim 6 , which is Escherichia coli.
8. The isolated host cell of claim 7 which is E. coli HB101 (pNTOM4) FERM BP-10369.
9. A vector comprising the DNA of claim 2 .
10. The vector of claim 9 , further comprising a DNA encoding a polypeptide having a glucose dehydrogenase activity.
11. The vector of claim 10 , wherein the polypeptide having a glucose dehydrogenase activity is a glucose dehydrogenase derived from Bacillus megaterium.
12. An isolated host cell transformed with the vector of claim 9 .
13. The isolated host cell of claim 12 , which is Escherichia coli.
14. A method of producing an optically active alcohol, which comprises reacting the isolated host cell of claim 6 or a treated product thereof, with a compound having a carbonyl group,
wherein the treated product is selected from the group consisting of a cell treated with a surfactant or an organic solvent, a dried cell, a disrupted cell, crude extract of the cell, and a fixed cell, and
wherein the carbonyl group is (3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanone, and the optically active alcohol is (2R, 3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol.
15. A method of producing an optically active alcohol, which comprises reacting the isolated host cell of claim 12 or a treated product thereof, with a compound having a carbonyl group,
wherein the treated product is selected from the group consisting of a cell treated with a surfactant or an organic solvent, a dried cell, a disrupted cell, crude extract of the cell, and a fixed cell, and
wherein the carbonyl group is (3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanone, and the optically active alcohol is (2R, 3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol.