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Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli

A technology of recombinant Escherichia coli and phospholipase, applied in the fields of genetic engineering and microbial fermentation

Active Publication Date: 2014-07-09
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional source of phospholipase A1 is animal pancreatic juice, but the extraction amount of this source is limited and expensive, and it has been gradually eliminated in recent years. Most of the phospholipase A1 is bound to the cell membrane, and it is difficult to carry out large-scale production. Therefore, it is important to study the cloning and expression of the phospholipase A1 gene in engineering bacteria and strive to improve its production. The main research directions in recent years

Method used

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  • Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli
  • Recombinant Escherichia coli, and method for preparing phospholipase A1 through using recombinant Escherichia coli

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Experimental program
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Embodiment 1

[0030] Design primers according to the gene sequence of NCBI GenBank: X66505.1, where the upstream primer is: 5′-CG GAATTC AGTATGTCTTTGAGTTTTACCTCTGC-3' (the underline indicates the EcoR Ⅰ restriction site), the downstream primer is: 5'-CC AAGCTT CGTTACTGCTGTCCGTATTGC-3' (the underline indicates the HindⅢ restriction site).

[0031] Phospholipase A was cloned by using the PCR method (primers as above) using Serratia liquefaction genomic DNA with the preservation number CICC No. 21538 as a template 1 The gene pla was connected with the vector pMD18T-simple to obtain the cloning vector pMD-pla. The cloning vector was transformed into Escherichia coli competent cell E.coli JM109, and positive clones were selected and verified by sequencing. Its base sequence is shown in SEQ ID NO: 1 showed that the results showed that phospholipase A 1 Gene cloning was successful. Sequence analysis showed that the gene sequence was identical to phospholipase A of GenBank number X66505.1 1 T...

Embodiment 2

[0033] The recombinant vector pMD-pla and the expression vector pET-28a (+) were double digested with EcoR Ⅰ and Hind Ⅲ. The enzyme digestion reaction system was 50 μL: vector 20 μL, enzyme reaction buffer 5 μL, EcoR Ⅰ 2.5 μL, Hind Ⅲ 2.5 μL, supplemented Double-distilled deionized water to 50 μL, reacted at 37°C for 2 hours. Recover the digested target gene and carrier DNA, and connect them with T4 DNA ligase, ligation reaction system 10 μL: target gene 2 μL, carrier DNA 1 μL, 10×T4 ligase buffer 1 μL, T4 DNA ligase 1 μL, double distilled deionized water 5 μL , reacted at 16°C for 12 hours.

[0034] Transform the ligation product into Escherichia coli competent cells E.coli JM109, the transformation method is as follows:

[0035] (1) Take 100 μL of E.coli JM109 and place it in a sterile 1.5ml EP tube;

[0036] (2) Add the above ligation product and mix gently;

[0037] (3) Put the above EP tube in a metal bath at 42°C, accurately time it for 90 seconds, and do not shake the...

Embodiment 3

[0043] Transform the constructed recombinant vector pET-pla into Escherichia coli BL21 (DE3), the transformation method is as follows:

[0044] (1) Take 100 μL of E.coli BL21 (DE3) and place it in a sterile 1.5ml EP tube;

[0045] (2) Add the above recombinant plasmid pET-pla and mix gently;

[0046] (3) Put the above EP tube in a metal bath at 42°C, accurately time it for 90 seconds, and do not shake the EP tube;

[0047] (4) Transfer the EP tube to an ice bath and cool for 5-10 minutes;

[0048] (5) Add 800 μL of sterile SOC medium to each tube, and place in a 37°C incubator to recover for 1 hour;

[0049] (6) Centrifuge at 8000r / min for 2min, suck off 800μL of supernatant medium, gently blow and suck the remaining medium and cells with a pipette gun, and transfer to LB solid containing final concentration of 50μg / ml kanamycin Plate, spread evenly and incubate in a 37°C incubator for 16-24 hours;

[0050] (7) Pick the transformant and verify the successful construction o...

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Abstract

The invention provides a strain of recombinant Escherichia coli and a method for preparing phospholipase A1 by using the strain. The present invention successfully constructed a strain of phospholipase A1 gene derived from the gene recombinant Escherichia coli BL21 (DE3) / pET-pla CCTCC M 2012423 of Serratia liquefaction CICC No. 21538, and used the strain to prepare lysophospholipids by liquid fermentation Enzyme A1, its preparation method includes slant culture, seed culture, spontaneous induction culture (including adding osmotic substances) and extraction of crude enzyme solution. Using the strain of the present invention to prepare phospholipase A1 through liquid fermentation has the advantages of high enzyme production and enzyme activity, simple production process, short production cycle, low cost, easy process amplification, etc., and has broad applications in the fields of oil refining and phospholipid modification. Application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and in particular relates to a strain of recombinant Escherichia coli and the preparation of phospholipase A by self-induced fermentation of the strain 1 Methods. Background technique [0002] Phospholipase A 1 (PhospholipaseA 1 , EC 3.1.1.32, referred to as PLA 1 ) belongs to hydrolase, which can specifically hydrolyze the Sn-1 acyl group of natural phospholipids to obtain Sn-2 acyl lysophospholipids and free fatty acids. Phospholipase A 1 It is an excellent surfactant and is widely used in the fields of food, medicine, feed and leather; in addition, phospholipase A 1 It can also be applied to vegetable oil degumming. Compared with traditional degumming methods, phospholipase A 1 Degumming, a new type of degumming method, has the advantages of mild reaction conditions, less by-products, energy saving and environmental protection, and has been widely p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/18C12R1/19C12R1/425
Inventor 张梁石贵阳延晋雷丁重阳顾正华
Owner JIANGNAN UNIV
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