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Phospholipase A2 mutant and preparation method thereof

A technology of mutant and phospholipase, applied in the field of genetic engineering of enzymes, can solve the problems of narrow enzyme source, complicated process, unfavorable mass production and utilization of phospholipase A, etc., and achieve the effect of reducing production cost and simplifying production links.

Active Publication Date: 2014-09-03
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although phospholipase A 2 It has a lot of application value, but there is a big gap from the needs of industrial production, especially compared with other enzyme production and application. The main reasons are: 1. The enzyme source is narrow. At present, phospholipase A 2 It is mainly extracted from animal pancreas, snake venom, and bee venom, and phospholipase A is also extracted from animal viscera or microorganisms 2 , 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 catalyzing the hydrolysis of phosphodiester bond at the sn-2 position of phospholipids. Bond base exchange is poorly studied

Method used

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  • Phospholipase A2 mutant and preparation method thereof
  • Phospholipase A2 mutant and preparation method thereof
  • Phospholipase A2 mutant and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Wild-type phospholipase A 2 Acquisition of the mature peptide gene

[0053] 1. Wild-type phospholipase A 2 The mature peptide gene comes from Streptomyces coelicolor ATCC23899, and its genomic DNA is extracted.

[0054] Wherein the extraction steps of Streptomyces coelicolor genomic DNA are as follows:

[0055] (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.

[0056] (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.

[0057] (3) Add 50 μL of 50 mg / mL lysozyme and digest at 4°C for 1 hour.

[0058] (4) Add 1 / 2 volume of 2% SDS solution and react for 10 minutes until the bacterial suspension becomes viscous.

[0059] (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

[0069] Embodiment 2: high activity phospholipase A 2 Gene acquisition.

[0070] 1. Wild-type phospholipase A 2 The gene is ligated into the T vector.

[0071] The purified target gene was connected to the pUC-T vector, and the recombinant plasmid was transformed into Escherichia coli DH5α, which was verified by EcoRI and MluI double enzyme digestion, and the wild-type phospholipase A 2 The gene has been successfully cloned into the T vector.

[0072] 2. Site-directed mutation

[0073] Based on overlapping PCR technology (see figure 2 ) for site-directed mutagenesis to construct a highly active phospholipase A 2 , design primers as follows:

[0074] Upstream P1 (SEQ ID NO.1): 5'-GCCCCCGCGGACAAGCCCCAGGT-3'

[0075] Downstream P2 (SEQ ID NO.2): 5'-TCAGCCGAAGATCTTGACGGC-3'

[0076] Overlapping primer P3 (SEQ ID NO.3): 5'-GGCCGCCTACGCGTTCGACTGGT-3'

[0077] Overlapping primer P4 (SEQ ID NO.4): 5'-ACCAGTCGAACGCGTAGGCGGCC-3'

[0078] Overlapping primer P5 (SEQ ID NO.5): 5'...

Embodiment 3

[0106] Embodiment 3: Bacillus subtilis high activity phospholipase A 2 Construction of recombinant bacteria

[0107] 1. Construction of expression vector pBSA43

[0108] 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.

[0109] 2. High activity phospholipase A 2 Expression vector pBSA43-plA 2 construction of m

[0110] The high-activity phospholipase A obtained through overlapping PCR construction 2 After the gene was digested with BamHI and HindIII, it was ligated with the same double digested Bacillus subtilis expressi...

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Abstract

The invention relates to a phospholipase A2 mutant and a preparation method thereof. The technical scheme is as follows: the preparation method comprises the following steps: carrying out site-specific mutagenesis on the amino acid residues of Glu37 and Asp78 of wild type phospholipase A2 derived from streptomyces coelicolor by utilizing a recombinant DNA technology to obtain a phospholipase A2 gene with higher activity; then expressing the phospholipase A2 gene with higher activity in a bacillus subtilis expression system and a pichia pastoris expression system (which comprises a pichia pastoris free expression system and a pichia pastoris cell surface display system) to obtain a recombinant strain capable of generating high-activity phospholipase A2; after the phospholipase A2 gene with higher activity is expressed, detecting that the enzyme activity of the high-activity phospholipase A2 is enhanced by 13.7% compared with that of the wild type phospholipase A2, wherein the enzyme activity of the phospholipase A2 can be up to 53.5 U / ml after the bacillus subtilis high-activity phospholipase A2 recombinant bacteria is fermented, the enzyme activity of pichia pastoris free-expression high-activity phospholipase A2 recombinant bacteria can be up to 106.4 U / ml, and the enzyme activity of a pichia pastoris cell surface display high-activity phospholipase A2 whole-cell catalyst can be up to 260 U / (g.stem cell).

Description

technical field [0001] The invention relates to a phospholipase and a preparation method thereof. The invention relates to phospholipase A with improved specific enzyme activity constructed by in vitro directed evolution of overlapping PCR technology 2 Mutants, specifically related to the use of molecular biology techniques to obtain phospholipase A with increased specific enzyme activity 2 A mutant belongs to the technical field of genetic engineering of enzymes. Background technique [0002] Phospholipase A 2 (Phospholipase A 2 , PLA 2 ), is a phospholipid-2-acyl hydrolase (EC3.1.1.4), which catalyzes the hydrolysis of the sn-2 ester bond of glycerophospholipids to generate lysophospholipids and fatty acids, which play an important role in the catabolism of phospholipids. In addition, some Phospholipase A 2 It has the function of catalyzing the base exchange of the phosphodiester bond at the sn-2 position of phospholipids. [0003] Phospholipase A 2 Widely present ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/75C12N1/21C12N15/81C12N1/19C12R1/125C12R1/84
CPCC12N9/18C12Y301/01004
Inventor 路福平刘逸寒张涛刘晓光王正祥王春霞王建玲
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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