Phospholipase d mutant for high-efficiency synthesis of phosphatidylserine and product and application thereof

By performing site-directed mutagenesis on phospholipase D to improve its thermal stability and catalytic activity, the problem of low production efficiency of phosphatidylserine in existing technologies has been solved, enabling the efficient preparation of phosphatidylserine products for the purpose of alleviating metabolic syndrome induced by antipsychotic drugs.

CN121271832BActive Publication Date: 2026-06-09ECA HEALTHCARE INC
View PDF 0 Cites 0 Cited by

Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
ECA HEALTHCARE INC
Filing Date
2025-11-19
Publication Date
2026-06-09

AI Technical Summary

Technical Problem

In the existing technology, the industrial production of phosphatidylserine (PS) is difficult due to problems such as insufficient catalytic efficiency and limited substrate specificity of natural phospholipase D, and there is a lack of effective intervention for metabolic syndrome caused by the antipsychotic drug olanzapine.

Method used

By performing site-directed mutagenesis on phospholipase D, particularly modifying the amino acid positions at 87, 136, 189, 379, and 458, mutations such as L87E, M136G, I189R, E379L, and A458Y were formed, improving its thermal stability and catalytic activity. Crude enzyme solution of mutant phospholipase D was then prepared by combining it with recombinant expression vectors and host cells.

Benefits of technology

The thermostability of mutant phospholipase D was improved from 45℃ to 55℃, and the relative enzyme activity increased from 15.8 U/mg to 37.3 U/mg. The phosphatidylserine series products prepared by the catalysis can effectively alleviate lipid metabolism disorders induced by antipsychotic drugs, providing clinical application and market value.

✦ Generated by Eureka AI based on patent content.

Smart Images

  • Figure CN121271832B_ABST
    Figure CN121271832B_ABST
Patent Text Reader

Abstract

This invention provides a mutant phospholipase D for the efficient synthesis of phosphatidylserine, its products, and applications. The mutant is based on phospholipase D with the amino acid sequence shown in SEQ ID NO.1, obtained by mutating one or more of five sites at positions 87, 136, 189, 379, and 458 of this amino acid sequence. The amino acid mutation types at these sites include: L87E, M136G, I189R, E379L, and A458Y. This invention provides both a mutant phospholipase D and its applications. This mutant phospholipase D exhibits improved thermostability and catalytic activity, enabling the catalytic preparation of phosphatidylserine series products. The phosphatidylserine series products prepared using this enzyme can effectively alleviate metabolic syndrome induced by antipsychotic drugs.
Need to check novelty before this filing date? Find Prior Art

Claims

1. A phospholipase D mutant for efficient synthesis of phosphatidylserine, characterized in that, The phospholipase D mutant is a mutant phospholipase D obtained by mutating several sites in the amino acid sequence of phospholipase D as shown in SEQ ID NO.

1. The amino acid sequences of the resulting mutant phospholipase D are shown in SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, or SEQ ID NO.

29.

2. A gene encoding the amino acid sequence of the phospholipase D mutant as described in claim 1.

3. The gene encoding the amino acid sequence of the phospholipase D mutant as described in claim 2, characterized in that, The nucleotide sequence of the gene is obtained by mutating the nucleotide sequence encoding the amino acid sequence of phospholipase D as shown in SEQ ID NO.1; the nucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO.1 is shown in SEQ ID NO.

30.

4. A recombinant expression vector comprising the gene encoding the amino acid sequence of the phospholipase D mutant as described in claim 3.

5. A recombinant bacterium comprising the recombinant expression vector as described in claim 4.

6. A method for preparing a phospholipase D mutant using recombinant bacteria containing a recombinant expression vector as described in claim 5, characterized in that, The method involves inoculating the recombinant bacteria into a culture medium, culturing overnight, centrifuging to collect the bacterial cells, and then concentrating the obtained bacterial cells by ultrafiltration to obtain a crude enzyme solution of mutant phospholipase D.

7. The method for preparing the phospholipase D mutant according to claim 6, characterized in that, The mutant phospholipase D crude enzyme solution is prepared by inoculating the recombinant bacteria into a liquid culture medium containing kanamycin and culturing it in a shaker at 37°C and 150 rpm for 16 h.

8. The application of a phospholipase D mutant prepared according to the method of claim 7, characterized in that, The phospholipase D mutant obtained by the method is used to prepare a series of phosphatidylserine products; the series of phosphatidylserine products are any one or more of soybean phosphatidylserine, phosphatidylserine rich in docosahexaenoic acid, and phosphatidylserine rich in eicosapentaenoic acid.