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

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. ...

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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 plasmid is not high, so the recombinant strain is not stable enough;

[0031] 3. In the gene cloning strategy, the probes used are several sets of artificially synthesized nucleotide fragments, all of which are less than 30 bp in length, and use such small nucleotide fragments as probes to catch phospholipase A from the genome library 2 Gene, the specificity is not strong, so the signal is weak during hybridization screening

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