Engineering bacterium for producing Phospholipase A2 (PLA2) and applications thereof

A technology for producing phospholipids and engineering bacteria, applied in the field of bioengineering, can solve the problems of low stability of plasmids, insufficient stability of recombinant strains, cumbersome operation, etc., and achieve the effect of separation and purification

Inactive Publication Date: 2011-10-26
HUAZHONG AGRI UNIV
View PDF0 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0029] 1. The enzyme production ability of the strain is still not ideal;
[0030] 2. In the construction of the genomic library, the vector used is the plasmid pUC118, which can only be replicated in Escherichia coli and cannot be replicated in Streptomyces. When constructing the expression strain, phospholipase A 2 Cloning the gene into Streptomyces plasmid pIJ680 is cumbersome to operate, and the stability of the plas

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Engineering bacterium for producing Phospholipase A2 (PLA2) and applications thereof
  • Engineering bacterium for producing Phospholipase A2 (PLA2) and applications thereof
  • Engineering bacterium for producing Phospholipase A2 (PLA2) and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] A phospholipase A 2 The preparation method of gene and leader signal peptide plasmid, its step is:

[0074] A. Used to amplify phospholipase A 2 Synthesis of primers for genes:

[0075] Access http: / / www.ncbi.nlm.nih.gov / GenBank, based on Streptomyces coelicolor A3(2) phospholipase A 2 Characterization of the DNA sequence from which Streptomyces phospholipase A 2 gene, and accordingly designed two PCR primers Oligo1 (SEQ ID NO.03) and Oligo2 (SEQ ID NO.04) with a length of 21 base pairs, and the designed primers were synthesized by a biotechnology company. The sequence and properties are detailed in the table below.

[0076] The characteristics of table 2 primers D11 and D12

[0077]

[0078] B, extraction of Streptomyces violaceum 2917 total DNA:

[0079] Dissolve 500ml of S.violaceoruber 2917 mycelia in 10ml of 2mg / ml LRTE solution (25mmol / LTris-HCl, pH8.0, 100mmol / L EDTA, RNase 50ug / ml.), lyse at 37°C until the solution is clear and translucent ;Add proteas...

Embodiment 2

[0098] a phospholipase A 2 The preparation method of engineering bacterium, its step is:

[0099] A. Add 25mL YEME medium to a 250ml Erlenmeyer flask with a stainless steel spring, and add glycine to a final concentration of 0.5%;

[0100] B. Inoculate 0.1 mL of Streptomyces S.lividans 1326 spore suspension, culture in a shake flask at 30°C for 36-40 hours; collect the bacteria by centrifugation;

[0101] C. Wash twice with 10.3% (W / V) sucrose solution; add 5 mL of lysozyme solution to the mycelium precipitate, and enzymolyze it in a water bath at 30° C. for 30-60 minutes;

[0102] D. Blow and suck 4 times with a sterile pipette, and continue to incubate in a water bath at 30°C for 15 minutes; add 5mL P buffer, blow and suck again; transfer to a filter equipped with absorbent cotton and filter; centrifuge the filtrate at 3,000rpm for 10 minutes to obtain protoplast precipitation;

[0103] E. Wash the protoplast pellet with 5mL P buffer solution for 1-2 times, and finally su...

Embodiment 3

[0106] The enzyme production ability comparison of engineering strain Streptomyces lividans LH001 and other bacterial strains etc., its steps are:

[0107] A. Inoculate the engineering bacteria Streptomyces lividans LH 001, the starting bacterium Streptomyces lividans 2917, the host strain Streptomyces lividans lividans 1326, the recombinant strain Streptomyces lividans lividans LEE2265, etc. into 100mL YEME medium respectively, and cultivate them at 30°C for 72 hour, 5000 rpm / centrifugation for 10 minutes, and the supernatant was obtained as the crude enzyme solution.

[0108] B. Qualitative and quantitative determination of phospholipase A by gas chromatography 2 vitality:

[0109] Principle: Phospholipase A 2 It can catalyze the hydrolysis reaction of the C-2 ester bond of glycerophospholipids to generate lysophospholipids and fatty acids, and qualitatively and quantitatively determine phospholipase A by measuring the types and quantities of fatty acids generated by the e...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention discloses an engineering bacterium for producing a PL A2 and applications thereof. A preparation method of the PLA2 comprises the following steps: A, preparing a PLA2 gene: designing a primer PCR and amplifying a PLA2 gene fragment, and cloning a DNA fragment containing the PLA2 gene (a sequence is SEQ ID NO.2) with an amplified fragment plaT as a probe; B, constructing a recombinant plasmid: cloning a Bam H1 restricted fragment with the size of 3 kb which contains the PLA2 gene to a high-copy plasmid pHZ132 to obtain a recombinant plasmid pLH 001; and C, constructing the engineering bacterium: introducing the plasmid pLH001 into a streptomyces lividans 1326 of a host bacterium through protoplast transformation to obtain a streptomyces lividans LH001 of the engineering bacterium. The engineering bacterium has good stability and the introduced plasmid is not easy to lose. The secretory active PLA2 (an amino acid sequence is SEQ ID NO.1) can be generated through liquid fermentation, the enzymatic activity of a fermentation liquid is equal to or more than 2,000 U/mL, and the enzyme production capability is better than present levels. The PLA2 can be applied to fields of vegetable oil degumming, lysophospholipid preparation and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically relates to a kind of phospholipase A 2 engineering bacteria, involving a phospholipase A-containing 2 The gene plasmid also involves a phospholipase A producing 2 The preparation method of the engineering bacteria, the phospholipase A produced by the engineering bacteria fermentation 2 Application in vegetable oil degumming and preparation of lysophospholipids. Background technique [0002] Phospholipase A 2 (Phospholipase A 2 , PLA 2 ), phospholipid-2-acyl hydrolase (EC 3.1.1.4), is an enzyme that plays an important role in phospholipid catabolism. It catalyzes the hydrolysis reaction of the C-2 ester bond of glycerophospholipids to generate lysophospholipids and fatty acids. [0003] Phospholipase A 2 It was first isolated from liver tissue by Belfanti in 1924 by biochemical methods. Later, people isolated this enzyme from other tissues such as kidney and prostate. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N1/21C11B3/00C12P9/00C12R1/465
Inventor 李维琳
Owner HUAZHONG AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap