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A genetically engineered bacterium producing phospholipase d and its construction method and application

A technology of genetically engineered bacteria and phospholipase, applied in genetic engineering, microorganism-based methods, applications, etc., can solve the problems of no PLD product, immature PLD, etc., and achieve low cost, convenient enzyme source and high yield. Effect

Active Publication Date: 2020-06-12
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on PLD in our country is still immature, and there is no related PLD product at present.

Method used

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  • A genetically engineered bacterium producing phospholipase d and its construction method and application
  • A genetically engineered bacterium producing phospholipase d and its construction method and application
  • A genetically engineered bacterium producing phospholipase d and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of recombinant Escherichia coli BL21(DE3)-pET22b-PLD.

[0036] (1) Using the genomic DNA of Streptomyces sp. (strain PMF) as a template to clone the phospholipase gene, the base sequence of which is shown in SEQ ID NO.2;

[0037] (2) Cloning the phospholipase D gene obtained in (1) into the pET-22b(+) expression vector to construct a recombinant expression plasmid;

[0038] (3) The recombinant plasmid obtained in (2) was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant Escherichia coli BL21(DE3)-pET22b-PLD1.

Embodiment 2

[0040] The composition of the seed medium is: NaCl 5g / L, peptone 10g / L, yeast powder 5g / L, pH 7.4, sterilized at 121°C for 20min.

[0041] The components of the fermentation medium are: peptone 10g / L, yeast extract 5g / L, NaCl 5g / L, sterilized at 121°C for 20min.

[0042] Inoculate the recombinant Escherichia coli BL21(DE3)-pET22b-PLD1 in the seed medium containing 50mg / mL ampicillin, and culture it in the shake flask at 37°C and 200r / min until the logarithmic growth phase, as the seed liquid; then the seed liquid Inoculate into 100mL fermentation medium containing ampicillin 50mg / mL according to 5% inoculum amount, and culture in shake flask at 37°C and 200r / min until OD 600 =0.6, then add IPTG to a final concentration of 0.05mM, induce culture at 30°C, 200r / min for 12h; centrifuge the fermentation broth at 4°C, 8000r / min for 10min, and take the supernatant to obtain a crude phospholipase D enzyme solution.

[0043]Dissolve lecithin in dichloromethane at a concentration of 10...

Embodiment 3

[0046] Embodiment 3: Phospholipase D gene codon optimization

[0047] (1) Escherichia coli codon optimization was performed on the phospholipase D gene derived from Streptomyces sp. (strain PMF) to obtain the optimized phospholipase D gene, whose sequence is shown in SEQ ID NO.1:

[0048] (2) Cloning the phospholipase D gene obtained in (1) into the pET-22b(+) expression vector to construct a recombinant expression plasmid;

[0049] (3) The recombinant plasmid obtained in (2) was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant Escherichia coli BL21(DE3)-pET22b-PLD2.

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Abstract

The invention discloses a gene sequence for coding phospholipase D, which belongs to the fields of biotechnology and preparation of phospholipids products. The gene sequence of the phospholipase D is sourced from Streptomyces sp.(strain PMF), and the gene sequence capable of being expressed in escherichia coli is obtained by virtue of the optimization of a codon. The invention also constructs a genetically engineered bacterium for producing phospholipase D, the phospholipase D produced by utilizing the recombinant Escherichia coli is used as a catalyst to produce phosphatidylserine, the enzyme is convenient in source, the yield is high, the fermentation process is simple, the cost is low, and the industrialization prospect is good.

Description

technical field [0001] The invention belongs to the field of biotechnology and preparation of phospholipid products, and specifically relates to a genetically engineered bacterium producing phospholipase D and its construction method and application. Background technique [0002] Phospholipase D (PLD), as a member of the phosphodiesterase superfamily, is one of the key enzymes that catalyze the first step in the hydrolysis of phospholipids. It is an important class of transmembrane signal transduction enzymes, belonging to Extracellular enzymes, encoded by different members of a gene family, are widely found in microorganisms, plants and animals. It can not only hydrolyze phosphatidylcholine to generate choline and phosphatidic acid, but also catalyze the transphospholipid reaction, transferring the hydroxyl group of the primary alcohol on the lipid chain to the phosphatidyl part of the phosphatidic acid product. The phosphoryl group transfer function of PLD is particularly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/16C12N15/70C12P7/6481C12P13/06C12Y301/04004
Inventor 陈可泉庞洋王昕
Owner NANJING TECH UNIV
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