Oleic acid hydratase OhyA1 mutant as well as preparation method and application thereof

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

Application Number
CN202610664465.X
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
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

The invention provides an oleic acid hydratase OhyA1 mutant as well as a preparation method and application thereof, and belongs to the technical field of biological catalysis and biochemical engineering. The amino acid sequence of the oleic acid hydratase OhyA1 mutant provided by the invention is as shown in SEQ ID No. 1. Compared with a parent enzyme, the oleic acid hydratase OhyA1 mutant has the advantages that due to deletion of phenylalanine side chain benzene ring groups at two sites, an active center space structure is reconstructed, the volume of a substrate binding pocket is enlarged, and the catalytic efficiency of a long-chain fatty acid substrate (especially linoleic acid) containing multiple double bonds is remarkably improved. The oleic acid hydratase OhyA1 mutant provided by the invention has high specificity to oleic acid and only catalyzes a cis-9 double bond hydration reaction of oleic acid, the single product is 10-hydroxy fatty acid, isomer generation is avoided, the product purity can reach 95% or above, the oleic acid conversion rate can reach 85% or above, and the oleic acid hydratase OhyA1 mutant is remarkably superior to an existing microbial conversion system.
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