Hydrolase and method for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid using same
The present invention provides a novel hydrolase that can industrially produce optically highly pure (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid with high efficiency at low costs, and a production method using the hydrolase.
1. A method for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid, comprising bringing a hydrolase, a microorganism or cell having an ability to produce the hydrolase, a and/or a culture solution containing the hydrolase into contact with dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid represented by formula (I) to produce (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid represented by formula (II),
wherein the hydrolase comprises a polypeptide of the following (a), (b), or (c):
(a) a polypeptide consisting of an amino acid sequence resulting from the deletion, insertion, substitution, and/or addition of 1 to 50 amino acids in the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in the formula (1):
wherein R is an alkyl group having 1 to 6 carbon atoms, and a ratio of Syn isomer to Anti isomer in formula (II) is equal to or less than 0.2%;
(b) a polypeptide consisting of an amino acid sequence with not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in formula (1);
(c) a polypeptide consisting of an amino acid sequence with not less than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, wherein the amino acid sequence comprises:
(i) 5 consecutive residues DATRG corresponding to residues 75-79 of SEQ ID NO: 4 and residues 80-84 of SEQ ID NO: 6;
ii 4 consecutive residues PYGF corresponding to residues 132-135 of SEQ ID NO: 4 and residues 136-139 of SEQ ID NO: 6;
iii 4 consecutive residues NWPG corresponding to residues 222-225 of SEQ ID NO: 4 and residues 226-229 of SEQ ID NO: 6; and/or
(iv) 5 consecutive residues PGWPG corresponding to residues 285-289 of SEQ ID NO: 4 and residues 289-293 of SEQ ID NO: 6.
2. The production method according to claim 1 , wherein R in the formula (1) is an ethyl group.
3. The production method according to claim 1 , wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid encoding the hydrolase comprising the polypeptide of following (a), (b), or (c).
4. The production method according to claim 1 , wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid comprising a base sequence of the following (d), (e), (f), or (g):
(d) the base sequence shown in SEQ ID NO: 3 or 5;
(e) a nucleic acid consisting of a base sequence resulting from the substitution, deletion, and/or addition of 1 to 150 bases in the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in formula (1);
wherein R is an alkyl group having 1 to 6 carbon atoms in formula (1), and wherein the ratio of Syn isomer to Anti isomer in formula (II) is equal to or less than 0.2%;
(f) a nucleic acid consisting of a base sequence having not less than-60% of 90% sequence identity with the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in formula (1);
(g) a nucleic acid having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 or 5 under the condition of washing once at 60° C., 1×SSC, and 0.1% SDS, and encoding a polypeptide having an activity to catalyze the reaction shown informula (1).
5. A method for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid, comprising bringing a treated product of a microorganism or cell having the ability to produce a hydrolase into contact with dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid represented by formula (I) to produce (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid represented by formula (II),
wherein the hydrolase comprises a polypeptide of the following (a), (b), or (c):
(a) a polypeptide consisting of an amino acid sequence resulting from the deletion, insertion, substitution, and/or addition of 1 to 50 amino acids in the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in formula (1):
wherein R is an alkyl group having 1 to 6 carbon atoms, and a ratio of Syn isomer to Anti isomer in formula (II) is equal to or less than 0.2%;
(b) a polypeptide consisting of an amino acid sequence with not less than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, and an activity to catalyze the reaction shown in formula (1);
(c) a polypeptide consisting of an amino acid sequence with not less than 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 6, wherein the amino acid sequence comprises:
(i) 5 consecutive residues DATRG corresponding to residues 75-79 of SEQ ID NO: 4 and residues 80-84 of SEQ ID NO: 6;
(ii) 4 consecutive residues PYGF corresponding to residues 132-135 of SEQ ID NO: 4 and residues 136-139 of SEQ ID NO: 6;
(iii) 4 consecutive residues NWPG corresponding to residues 222-225 of SEQ ID NO: 4 and residues 226-229 of SEQ ID NO: 6; and/or
(iv) 5 consecutive residues PGWPG corresponding to residues 285-289 of SEQ ID NO: 4 and residues 289-293 of SEQ ID NO: 6.
6. The production method according to claim 5 , wherein R in the formula (1) is an ethyl group.
7. The production method according to claim 5 , wherein the microorganism or cell is a microorganism or cell transformed with a nucleic acid comprising a base sequence of the following (d), (e), (f), or (g):
(d) the base sequence shown in SEQ ID NO: 3 or 5;
(e) a nucleic acid consisting of a base sequence resulting from the substitution, deletion, and/or addition of 1 to 150 bases in the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in formula (1):
wherein R is an alkyl group having 1 to 6 carbon atoms in formula (1), and the ratio of Syn isomer to Anti isomer in formula (II) is equal to or less than 0.2%;
(f) a nucleic acid consisting of a base sequence having not less than 90% of sequence identity with the base sequence shown in SEQ ID NO: 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in formula (1);
(g) a nucleic acid having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 or 5 under the condition of washing once at 60° C., 1×SSC, and 0.1% SDS, and encoding a polypeptide having an activity to catalyze the reaction shown in formula (1).