Recombinant escherichia coli and method for preparing phospholipase A1 by same

A technology of recombinant Escherichia coli and phospholipase, applied in the field of genetic engineering and microbial fermentation, can solve the problems of limited extraction, late start, and low yield

Inactive Publication Date: 2013-04-17
JIANGNAN UNIV
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  • Summary
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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
Phospholipase A produced by microorganisms at home and abroad 1 It started late, and the yield is generally low: in 1988, Michael Givskova et al. extracted phospholipase A from Serratia liquefaction 1 The gene was cloned and expressed in Escherichia coli, and its enzyme activity was less than 1U / ml (Givskov M, Molin S.Secretion of Serratia liquefaciens phospholipase from Eseherichia.coli[J].Mol.Microbiol, 1993(8):229-42); In 2000, M. Hartmann et al. used random chemical mutation, single cell fusion and gene hybridization techniques to screen out 4 mutant strains of Tetrahymena thermophila, with the highest enzyme activity of 35.2±2.60U / ml (Hartmann M, et al. Screening for and charaeterization of phospholipase A 1 hypersecretory mutants of Tetrahymena thermophila[J].Appl.Microbiol.Biotechnol, 2000(54):390-396); Phospholipase A 1 The strain xjF1, after preliminary optimization, has the highest enzyme activity of 17.5U / ml (Fu Jianhong, Tang Huigui, Yao Bin, etc. A strain producing low-temperature alkaline phospholipase A 1 Screening of cold-resistant bacteria and preliminary study on fermentation conditions. Industrial Microbiology 2008.38: 12-16)

Method used

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Embodiment 1

[0029] Example 1 Phospholipase A 1 Gene cloning

[0030] Design primers according to the gene sequence numbered ATCC 29220 provided by NCBI GenBank database, where the upstream primer is: 5’-G GAATTC GATGCGGGCGATTCTG-3' (underline indicates EcoRI restriction site), the downstream primer is: 5'- CGAGCTC TCAGGTCGCTGCGATGTC-3' (underline indicates HindⅢ restriction site).

[0031] The genomic DNA of Citrobacter youngseri with the deposit number CICC No. 21596 was used as a template, and the phospholipase A was cloned by PCR (primers mentioned above) 1 Gene phla, and ligated with the vector pMD18T-simple to obtain the cloning vector pMD-phla. The cloning vector was transformed into E. coli competent cells E. coli JM109. Positive clones were selected and sequenced for verification. The base sequence is shown in SEQ ID NO:1 Show, the results show that phospholipase A 1 Gene cloning was successful. Sequence analysis showed that the gene sequence is similar to the above-mentioned phosph...

Embodiment 2

[0032] Example 2 Construction of recombinant vector pET-phla

[0033] The recombinant vector pMD-phla and the expression vector pET-28a(+) were double digested with EcoR I and HindⅢ. The digestion reaction system was 50μL: vector 20μL, enzyme reaction buffer 5μL, EcoR I 2.5μL, HindⅢ2.5μL, supplement Double-evaporate deionized water to 50μL and react at 37°C for 2 hours. Recover the digested target gene and vector DNA and ligate with T4 DNA ligase. The ligation reaction system 10μL: target gene 2μL, carrier DNA 1μL, 10×T4 ligase Buffer 1μL, T4 DNA ligase 1μL, double distilled to deionize Water 5μL, react at 16°C for 12h.

[0034] The ligation product was transformed into E.coli JM109 competent cell, the transformation method is as follows:

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

[0036] (2) Add the above-mentioned connection product and mix gently;

[0037] (3) Put the above EP tube in a 42℃ metal bath, time accurately for 90s, do not shake th...

Embodiment 3

[0042] Example 3 Construction of recombinant E. coli BL21(DE3) / pET-phla CCTCC M 2012424

[0043] The constructed recombinant vector pET-phla is transformed into E. coli BL21 (DE3), the transformation method is as follows:

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

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

[0046] (3) Put the above EP tube in a 42℃ metal bath, time accurately for 90s, 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 800μL of sterile SOC medium to each tube and place it in a 37°C incubator for recovery for 1 hour;

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

[0050] (7) Pick the transformants, and...

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Abstract

The invention provides recombinant escherichia coli and a method for preparing phospholipase A1 by the recombinant escherichia coli. The invention successfully constructs a strain of genetic recombinant escherichia coli BL21(DE3)/pET-ph1a CCTCC (China Center For Type Culture Collection) No:M2012424 of which a phospholipase A1 gene is from Citrobacter youngae CICC (China Center of Industrial Culture Collection) No.21596. The strain is subjected to liquid fermentation to prepare the hemolytic phospholipase A1. The preparation method of the hemolytic phospholipase A1 comprises the steps of slant culture, seed culture, liquid fermentation culture and extraction of a crude enzyme. The phospholipase A1 is prepared by utilizing the strain disclosed by the invention through liquid fermentation; and the recombinant escherichia coli has the advantages of high enzyme yield and enzyme activity, simple production process, short production period, low cost, easiness for process amplification and the like and lays a foundation for scale production of the phospholipase A1.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and specifically relates to a recombinant E. coli strain and liquid fermentation using the strain to prepare phospholipase A 1 Methods. Background technique [0002] Phospholipase A 1 (Phospholipase A 1 , EC 3.1.1.32, referred to as PLA 1 ) Is a hydrolase that can specifically hydrolyze the Sn-1 acyl group of natural phospholipids to obtain Sn-2 acyl lysophospholipids and free fatty acids. Phospholipase A1 is an excellent surfactant, which is widely used in food, medicine, feed, leather and other fields; in addition, phospholipase A 1 It can also be used for vegetable oil degumming. Compared with traditional degumming methods, phospholipase A 1 This new type of degumming method has the advantages of mild reaction conditions, less by-products, energy saving and environmental protection, and has been widely promoted in the oil refining industry in recent years. [00...

Claims

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Application Information

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