Method for preparing phosphatidylserine with docosahexaenoic acid at sn-2 bit

A phosphatidylserine and serine technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of unfavorable production and utilization of phospholipase A, narrow enzyme source, and little research

Active Publication Date: 2014-08-27
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Phospholipase A 2 It has high application value, but there are still relatively few studies on its application technology. The main reasons are: 1. The enzyme source is narrow. At present, phospholipase A2 is mainly extracted from animal pancreas, snake venom, bee venom, and also from animal viscera. Or extract phospholipase A2 from microorganisms, but the process is complicated, which is not conducive to phospholipase A 2 2. The mechanism of action of this enzyme has not been fully clarified. At present, there are many studies on its catalysis of the hydrolysis of the 2-position phosphodiesterate bond of the phospholipid glycerol Bonded base exchange is poorly studied

Method used

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  • Method for preparing phosphatidylserine with docosahexaenoic acid at sn-2 bit
  • Method for preparing phosphatidylserine with docosahexaenoic acid at sn-2 bit
  • Method for preparing phosphatidylserine with docosahexaenoic acid at sn-2 bit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Wild-type phospholipase A 2 Acquisition of the mature peptide gene

[0070] 1. Wild-type phospholipase A 2 The mature peptide gene comes from Streptomyces coelicolor ATCC23899, and its genomic DNA is extracted.

[0071] Wherein the extraction steps of Streptomyces coelicolor genomic DNA are as follows:

[0072] (1) Pick a ring of bacteria from the culture plate and inoculate in 40mL of appropriate medium, culture at 26°C, 150r / min for 2-3d.

[0073](2) Take 1 mL of the culture solution in a 1.5 mL EP tube, centrifuge at 12,000 r / min for 10 min, pour off the supernatant, and resuspend with 200 μL of Solution I.

[0074] (3) Add 50 μL of 50 mg / mL lysozyme and digest at 4°C for 1 hour.

[0075] (4) Add 1 / 2 volume of 2% SDS solution and react for 10 minutes until the bacterial suspension becomes viscous.

[0076] (5) Add an equal volume of saturated phenol: chloroform = 1:1, mix well, centrifuge for 10 min, transfer the supernatant to another clean EP tube, ...

Embodiment 2

[0086] Embodiment 2: high activity phospholipase A 2 Gene acquisition.

[0087] 1. Wild-type phospholipase A 2 The gene was ligated into the vector pUC-T.

[0088] Purified target gene plA 2 Ligated with vector pUC-T to form recombinant plasmid pUC-T-plA 2 , The recombinant plasmid was transformed into Escherichia coli DH5α.

[0089] 2. Site-directed mutation

[0090] Based on overlapping PCR technology (see figure 2 ) for site-directed mutagenesis to construct a highly active phospholipase A 2 , design primers as follows:

[0091] Upstream P1 (SEQ ID NO: 1): 5'-GCCCCCGCGGACAAGCCCCAGGT-3'

[0092] Downstream P2 (SEQ ID NO: 2): 5'-TCAGCCGAAGATCTTGACGGC-3'

[0093] Overlapping primer P3 (SEQ ID NO: 3): 5'-GGCCGCCTACGCGTTCGACTGGT-3'

[0094] Overlapping primer P4 (SEQ ID NO: 4): 5'-ACCAGTCGAACGCGTAGGCGGCC-3'

[0095] Overlapping primer P5 (SEQ ID NO: 5): 5'-GGGCAGCTTCCACGCCAACAAGA-3'

[0096] Overlapping primer P6 (SEQ ID NO: 6): 5'-TCTTGTTGGCGTGGAAGCTGCCC-3'

[009...

Embodiment 3

[0125] Embodiment 3: Bacillus subtilis high activity phospholipase A 2 and the construction of highly active phospholipase D recombinant bacteria

[0126] 1. Construction of expression vector pBSA43

[0127] pBSA43 is based on the Escherichia coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloned into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium. get. it comes with amp r Gene that can use ampicillin resistance as a selectable marker in E. coli. At the same time with Km r , Kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0128] 2. High activity phospholipase D and high activity phospholipase A 2 Expression vectors pBSA43-pldm and pBSA43-plA 2 construction of m

[0129] The high-activity phospholipase A obtained through overlapping PCR construction 2 Gene and high-activity phospholipa...

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Abstract

The invention relates to a method for preparing phosphatidylserine (2-DHA-PS) with docosahexaenoic acid at the sn-2 bit through high-activity phospholipase A2 and high-activity phospholipase D. Directed evolution is achieved through the overlapping PCR technology so that the high-activity phospholipase A2 and the high-activity phospholipase D can be obtained; the 2-DHA-PS is prepared through catalysis by means of the high-activity phospholipase A2 and the high-activity phospholipase D, phosphatidylserine is generated through phosphatidylcholine and serine under the catalysis of the high-activity phospholipase D first, and then the 2-DHA-PS is generated through the phosphatidylserine and the docosahexaenoic acid under the catalysis of the high-activity phospholipase A2. The relative content of the 2-DHA-PS in the product synthesized through the method is high, and the defects of an existing synthesizing method are effectively overcome.

Description

technical field [0001] The invention belongs to the field of biocatalysis, in particular to high activity phospholipase A 2 and a method for preparing phosphatidylserine (2-DHA-PS) whose sn-2 position is docosahexaenoic acid catalyzed by high-activity phospholipase D. Background technique [0002] Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid. The carbon chain structure of DHA contains six double bonds and is highly unsaturated. DHA is difficult to synthesize in the human body and needs to be provided by food. It is one of the essential fatty acids in the human body. DHA is closely related to the physiological functions of the human body. It can maintain the normal function and growth of the brain and retina. It has the functions of inhibiting platelet aggregation, antithrombotic, regulating blood lipids, improving immunity, strengthening the brain and improving intelligence. It is effective in inhibiting inflammation and some cancers. , The occurrence of diab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12N9/16C12N15/75C12N15/81C12N1/21C12N1/19C12R1/125C12R1/84
Inventor 路福平刘逸寒张涛刘晓光王正祥王春霞王建玲
Owner TIANJIN UNIV OF SCI & TECH
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