An oleic acid hydratase OhyA1 mutant, its preparation method and application

By performing site-directed mutagenesis on the oleic acid hydrase of Stenotrophomonas maltophilia OhyA1, the OhyA1-F94A/F205A mutant was formed, solving the problems of low purity and low efficiency in the preparation of hydroxy fatty acids in the existing technology. This resulted in the preparation of 10-hydroxy fatty acids with high efficiency and high purity, which is suitable for industrial applications.

CN122303208APending Publication Date: 2026-06-30BEIJING YANZHISHAN TECH CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
BEIJING YANZHISHAN TECH CO LTD
Filing Date
2026-05-14
Publication Date
2026-06-30

AI Technical Summary

Technical Problem

Existing chemical synthesis methods for preparing hydroxy fatty acids suffer from low purity, demanding and difficult-to-control reaction conditions, low efficiency of microbial transformation methods, low catalytic activity of oleic acid hydrase OhyA1 in heterologous expression systems, and poor substrate tolerance, making it difficult to meet industrial requirements.

Method used

By site-directed mutagenesis of Stenotrophomonas maltophilia OhyA1 oleic acid hydrase, replacing the phenylalanine residues at positions 94 and 205 with alanine, the OhyA1-F94A/F205A mutant was formed. This expanded the substrate binding pocket volume, improved the catalytic efficiency for long-chain fatty acids with multiple double bonds, and optimized the recombinant expression and purification process.

Benefits of technology

It achieves efficient and high-purity preparation of 10-hydroxy fatty acids, significantly improves catalytic efficiency, enhances substrate tolerance, is suitable for industrial production, and has mild reaction conditions, meeting the requirements of green chemistry.

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Abstract

This invention provides an oleic acid hydratase OhyA1 mutant, its preparation method, and its applications, belonging to the fields of biocatalysis and biochemical engineering. The amino acid sequence of the oleic acid hydratase OhyA1 mutant provided by this invention is shown in SEQ ID No. 1. Compared with the parent enzyme, the OhyA1 mutant oleic acid hydratase of this invention exhibits a restructured active site due to the deletion of phenylalanine side chain benzene ring groups at two sites, leading to an increased substrate binding pocket volume and significantly improved catalytic efficiency for long-chain fatty acid substrates containing multiple double bonds (especially linoleic acid). The OhyA1 mutant oleic acid hydratase provided by this invention exhibits high specificity for oleic acid, catalyzing only the cis-9 double bond hydration reaction of oleic acid. The product is a single 10-hydroxy fatty acid, avoiding isomer formation. The product purity can reach over 95%, and the oleic acid conversion rate can reach over 85%, significantly superior to existing microbial transformation systems.
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