Recombinant escherichia coli, method for preparing phospholipase C and application

A technology for recombining Escherichia coli and phospholipase, which is applied in the fields of enzyme genetic engineering and enzyme engineering, can solve problems such as non-application, and achieve the effects of low cost, high enzyme production and short production cycle.

Inactive Publication Date: 2014-01-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chen Tao’s team from Wuhan Institute of Virology has been committed to the research of microbial PLC. Li Caifeng and Zhan Yishu screened PLC-producing Vibrio harveii and Bacillus cereus, respectively. , Zhao Jinxing expressed Pseudomonas aeruginosa PLC using Escherichia coli, but none of the prepared PLC was applied to oil degumming

Method used

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  • Recombinant escherichia coli, method for preparing phospholipase C and application
  • Recombinant escherichia coli, method for preparing phospholipase C and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Cloning of embodiment 1 phospholipase C gene

[0040] Primers were designed according to the sequence of the plc gene of L.monocytogenes (YP005964174.1) provided by NCBI, where the upstream primers were:

[0041] 5'-CG GAATTC ATGTGTTGTGATGAATACTTACAAA-3' (the underlined part indicates the EcoR I restriction site);

[0042] Downstream primers are:

[0043] 5'-AT GCGGCCGC TTATTCATTTGTTTTTTT-3' (the underlined part indicates the NotI restriction site).

[0044] Using the genomic DNA of Listeria monocytogenes (L.monocytogenes) with the preservation number CICC No. 21540 as a template, the lysophospholipase C gene was cloned by PCR, and connected with the vector pMD18T-simple to construct a clone Vector pMD18T-simple-lm-plc, transform the cloning vector into Escherichia coli competent cells E.coli JM109, select positive clones, and sequence verification. The base sequence is shown in SEQ ID NO: 1, and the results show that the phospholipase C gene Cloning succeeded.

Embodiment 2

[0045] The construction of embodiment 2 recombinant plasmid pET-L.m-plc

[0046] The recombinant vector pMD18T-simple-lm-plc and the expression vector pET-28a (+) were double digested with EcoR I and Not I, and the enzyme digestion reaction system was 20 μL: plasmid 10 μL, Buffer 32 μL, Not I0.5 μL, EcoR I0. 5 μL, 100*Bsa 0.2 μL, H2O 6.8 μL. The target gene and carrier DNA were recovered and ligated with T4 DNA ligase. The ligation reaction system was 10 μL: 6 μL of target gene, 1.2 μL of carrier DNA, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and 0.8 μL of H200, and reacted at 16°C for 12 hours.

[0047] Transform the ligation product into Escherichia coli competent cells E.coli JM109, the transformation method is as follows:

[0048] (1) Add 10ul of the above ligation product to the E.coli JM109 competent cells (100ul / tube) that have been aliquoted, mix gently, and ice-bath for 30min;

[0049] (2) Put the above EP tube in a 42°C water bath, accurately time 90s, an...

Embodiment 3

[0055] Example 3 Construction of recombinant Escherichia coli BL21(DE3)-pET28a-L.m-PLC, deposit number CCTCC No.M2013301

[0056] Transform the constructed recombinant plasmid pET-L.m-plc into Escherichia coli BL21 (DE3), the transformation method is as follows:

[0057] (1) Add 0.5ul of the above-mentioned recombinant plasmid pET-lm-plc to 100μL of E.coli BL21 (DE3) competent cells (100ul / tube) that has been aliquoted, mix gently, and ice-bath for 30min;

[0058] (2) Put the above EP tube in a 42°C water bath, accurately time 90s, and do not shake the EP tube;

[0059] (3) Transfer the EP tube to an ice bath and cool for 3 minutes;

[0060] (4) Add 890 μL of sterile TB medium to each tube, place on a shaker at 37°C, 100r / min, and recover for 1h;

[0061] (5) 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 kanamycin at a final concentration of 1...

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Abstract

The invention discloses recombinant escherichia coli, a method for preparing phospholipase C and application. The recombinant escherichia coli BL21(DE-3)-pET-28a-L.m-phospholipase C (PLC) of which the target gene is from listeria monocyogenes (L.monocytogenes) is utilized as a fermentation strain to carry out liquid fermentation, so as to prepare phospholipase C. The method disclosed by the invention is simple in enzyme production process, short in production cycle and low in cost. A glycerophosphate bond on glyceryl phosphatide C3 can be specifically hydrolyzed to produce diglyceride (DAG) by using recombinant phospholipase C; plant crude oil (soybean oil, colleseed oil, rice bran oil and the like) is degummed by phospholipase C.

Description

technical field [0001] The invention relates to a production method of phospholipase C derived from Listeria monocytogenes and its application in the oil degumming industry, belonging to the fields of enzyme gene engineering and enzyme engineering. Background technique [0002] Listeria monocytogenes (L.monocytogenes) is an important food-borne pathogen, the growth temperature ranges from -1.5°C to 45°C, and the optimum temperature is 30°C to 37°C. Listeria monocytogenes is a typical cold-resistant bacterium that can grow and multiply in the cold room of ordinary refrigerators, and can still partially survive at -20°C, and the longer the food containing Listeria monocytogenes is refrigerated, the greater the danger ; and has strong heat resistance, milk pasteurization temperature can not kill it. Facultative anaerobic, often grows in a microaerophilic environment containing CO2; does not require high nutrition, can form biofilm on the surface of objects, is resistant to ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/16C11B3/00C12R1/19C12R1/01
Inventor 常明余榛榛刘睿杰金青哲王兴国刘元法
Owner JIANGNAN UNIV
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