Carbonyl reductase, nucleic acid encoding same, and method for producing optically active compound using same
The present invention provides a carbonyl reductase having the activity of reducing a carbonyl group-containing compound to convert the compound into an optically active compound, and a production method of an optically active compound using the enzyme. Specifically, a carbonyl reductase having one or more mutations in which the 54th aspartic acid, the 157th methionine, the 170th alanine, the 211th isoleucine, the 214th methionine, and the 249th methionine are each substituted by other specific amino acid in the amino acid sequence shown in SEQ ID NO: 1 or a homologue of the amino acid sequence, and a production method of an optically active compound using the same are provided.
1 . A carbonyl reductase variant comprising an amino acid sequence having at least 90% and less than 100% sequence identity to SEQ ID NO:1 and having carbonyl reductase activity, the variant comprising at least one or more mutation selected from the group of mutations consisting of:
(a) a mutation in which the 54th aspartic acid in SEQ ID NO: 1 is substituted by valine;
(b) a mutation in which the 157th methionine in SEQ ID NO: 1 is substituted by valine;
(c) a mutation in which the 170th alanine in SEQ ID NO: 1 is substituted by serine;
(d) a mutation in which the 211th isoleucine in SEQ ID NO: 1 is substituted by alanine or asparagine;
(e) a mutation in which the 214th methionine in SEQ ID NO: 1 is substituted by leucine; and
(f) a mutation in which the 249th methionine in SEQ ID NO: 1 is substituted by leucine.
2 . The carbonyl reductase variant according to claim 1 , comprising at least two mutations selected from the group of mutations.
3 . The carbonyl reductase variant according to claim 1 , further comprising (g) a mutation in which the 166th valine in SEQ ID NO: 1 is substituted by alanine.
4 . A method for producing an optically active compound represented by the following formula (IV):
wherein:
R is a hydrogen atom, an alkyl group, or an aryl group, and
is a substituent having an aromatic ring or a heterocycle, the method comprising:
asymmetrically reducing a carbonyl group-containing compound selected from the group consisting of compounds represented by the following formulas (I), (II) and (III):
in the above-mentioned formulas (I), (II) and (III), R is as defined above, and
is as defined above, by bringing the carbonyl group-containing compound into contact with an enzyme, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, or a culture medium containing the enzyme obtained by culturing the microorganism or cell:
wherein the enzyme is a carbonyl reductase variant comprising an amino acid sequence having at least 90% and less than 100% sequence identity to SEQ ID NO: 1 and having carbonyl reductase activity, the variant comprising at least one or more mutation selected from the group consisting of:
(a) a mutation in which the 54th aspartic acid in SEQ ID NO: 1 is substituted by valine;
(b) a mutation in which the 157th methionine in SEQ ID NO: 1 is substituted by valine;
(c) a mutation in which the 170th alanine in SEQ ID NO: 1 is substituted by serine;
(d) a mutation in which the 211th isoleucine in SEQ ID NO: 1 is substituted by alanine or asparagine;
(e) a mutation in which the 214th methionine in SEQ ID NO: 1 is substituted by leucine; and
(f) a mutation in which the 249th methionine in SEQ ID NO: 1 is substituted by leucine.
5 . The production method according to claim 4 , wherein the carbonyl group-containing compounds represented by the formula (II) and the formula (III) are respectively an optically active form represented by the following formula (II′):
and an optically active form represented by the following formula (III′):
wherein R and
are as defined above.
6 . The production method according to claim 4 , wherein the substituent
wherein
is a substituent having an aromatic ring or a heterocycle,
7 . The production method according to claim 6 , wherein the substituent
8 . The production method according to claim 4 , wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding said carbonyl reductase variant and comprising a nucleotide sequence having not less than 90% and less than 100% sequence identity to SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity.
9 . A composition comprising:
an enzyme, wherein the enzyme is the carbonyl reductase variant according to claim 1 ,
a microorganism or cell having the ability to produce the enzyme,
a processed product of the microorganism or cell, or
a culture solution containing the enzyme obtained by:
culturing the microorganism or cell, and
catalyzing a reaction using a carbonyl group-containing compound represented by formula (I), (II), or (III):
to produce an optically active compound represented by formula (IV):
wherein in formulas (I), (II), (III), and (IV):
R is a hydrogen atom, an alkyl group, or an aryl group, and
is a substituent having an aromatic ring and/or a heterocycle.
10 . The method according to claim 4 , wherein the carbonyl reductase variant comprises at least two mutations selected from the group of mutations.
11 . The method according to claim 4 , wherein the carbonyl reductase variant further comprises (g) a mutation in which the 166th valine in SEQ ID NO: 1 is substituted by alanine.