Novel hydrolase and method for producing (1s,2s)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid using same

Patent metadata

Field	Value
Publication number	US-2021024905-A1
Application number	US-201917043339-A
Country	US
Kind code	A1
Filing date	Mar 29, 2019
Priority date	Mar 30, 2018
Publication date	Jan 28, 2021
Grant date	—

Abstract

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.

First claim

1 . A hydrolase comprising a polypeptide of any of the following (a)-(e):
(a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 (b) a polypeptide having 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 1-6 carbon atoms
(c) a polypeptide having an amino acid sequence resulting from the deletion, insertion, substitution and/or addition of one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, and an activity to catalyze the reaction shown in the formula (1)
(d) a polypeptide having an amino acid sequence with not less than 60% of sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, and an activity to catalyze the reaction shown in the above-mentioned formula (1)
(e) a polypeptide having an amino acid sequence with not less than 60% of sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, wherein the aforementioned amino acid sequence comprises one or more motif sequences selected from the following motif sequences (i)-(iv)
(i) a sequence consisting of 5 consecutive residues (aspartic acid (D), alanine (A), threonine (T), arginine (R), glycine (G))
(ii) a sequence consisting of 4 consecutive residues (proline (P), tyrosine (Y), glycine (G), phenylalanine (F))
(iii) a sequence consisting of 4 consecutive residues (asparagine (N), tryptophan (W), proline (P), glycine (G))
(iv) a sequence consisting of 5 consecutive residues (proline (P), glycine (G), tryptophan (W), proline (P), glycine (G)).

2 . The hydrolase according to claim 1 , wherein R in the formula (1) is an ethyl group.

3 . A nucleic acid encoding hydrolase according to claim 1 or 2 .

4 . The nucleic acid according to claim 3 , wherein the aforementioned nucleic acid comprises a base sequence of the following (f), (g), (h) or (i):
(f) the base sequence shown in SEQ ID NO: 1, 3 or 5 (g) a nucleic acid having a base sequence resulting from the substitution, deletion, and/or addition of one or plural bases in the base sequence shown in SEQ ID NO: 1, 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in the formula (1)

wherein R is an alkyl group having 1-6 carbon atoms
(h) a nucleic acid having a base sequence having not less than 60% of sequence identity with the base sequence shown in SEQ ID NO: 1, 3 or 5, and encoding a polypeptide having an activity to catalyze the reaction shown in the above-mentioned formula (1)
(i) a nucleic acid having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 1, 3 or 5 under stringent conditions, and encoding a polypeptide having an activity to catalyze the reaction shown in the above-mentioned formula (1).

5 . A method for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid, comprising bringing the hydrolase according to claim 1 or 2 , a microorganism or cell having an ability to produce the aforementioned enzyme, a treated product of the aforementioned microorganism or cell, and/or a culture solution containing the aforementioned enzyme obtained by culturing the aforementioned microorganism or cell into contact with dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid represented by the formula (I) to produce (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropane carboxylic acid represented by the formula (II).

6 . The production method according to claim 5 , wherein the aforementioned microorganism or cell is a microorganism or cell transformed with the nucleic acid according to claim 3 or 4 .

7 . The recombinant vector comprising the nucleic acid according to claim 3 or 4 .

8 . A transformant comprising the recombinant vector according to claim 7 .

Assignees

• Api Corp

Inventors

• Yoshida Toyokazu
• Ishida Koichi
• Miyake Ryoma
• Iura Takanobu
• Kawabata Hiroshi

Classifications

• C12P41/005 Primary

  by esterification of carboxylic acid groups in the enantiomers or the inverse reaction · CPC title

• C12P7/62

  Carboxylic acid esters · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12P41/00

  Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture · CPC title

• C12Y301/01

  Carboxylic ester hydrolases (3.1.1) · CPC title