Carboxylesterase mutants and their use in the production of (r)-3-cyclohexene-1-carboxylic acid

By mutating specific amino acid sequences of carboxylesterase EstA1, a highly active and selective carboxylesterase mutant was constructed, solving the problem of poor stereoselectivity of natural carboxylesterases and realizing a green synthesis process for the efficient preparation of optically pure (R)-3-cyclohexene-1-carboxylic acid.

CN119842663BActive Publication Date: 2025-10-24ZHEJIANG UNIV OF TECH
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Patent Information

Application Number
CN202510122936.X
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2025-01-26
Publication Date
2025-10-24
Estimated Expiration
2045-01-26

AI Technical Summary

Technical Problem

In existing technologies, natural wild-type carboxylesterases have poor stereoselectivity and low catalytic activity, resulting in complex processes and low overall yields in the preparation of optically pure (R)-3-cyclohexene-1-carboxylic acid, which is difficult to meet the needs of industrial applications.

Method used

By performing single-point or combined mutations on specific amino acid sequences of carboxylesterase EstA1, a highly active and selective carboxylesterase mutant EstA1 was constructed for catalytic resolution of methyl (R,S)-3-cyclohexene-1-carboxylate to prepare (R)-3-cyclohexene-1-carboxylic acid. The catalytic conditions were optimized by combining recombinant vectors and engineered bacteria.

Benefits of technology

It achieves high catalytic activity and high stereoselectivity, mild reaction conditions, low production cost, significantly improves the preparation efficiency of (R)-3-cyclohexene-1-carboxylic acid, with an ee value of >99% for the product, short reaction time, low catalyst dosage, simplified steps, and environmental friendliness.

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Abstract

The application discloses a carboxyesterase mutant and application thereof in production of optically pure (R)-3-cyclohexene-1-carboxylic acid, wherein the carboxyesterase EstA1 mutant is obtained by single-point mutation or combined mutation of positions 70, 128, 133, 227, 248, 249, 297, 301, 326 or 329 of the amino acid sequence shown in SEQ ID No. 1. The application provides the carboxyesterase EstA1 mutant with high catalytic activity and high stereoselectivity to racemic (R,S)-3-cyclohexene-1-carboxylic acid methyl ester, and the obtained mutant has the characteristics of mild reaction condition, high enantiomer selectivity, short reaction time, small amount of catalyst, simple reaction system and environmental friendliness in the process of enantioselective resolution of (R)-3-cyclohexene-1-carboxylic acid.
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