Recombinant expression and application of lactobacillus casei phospholipase A2 gene

A technology of cheese milk and bacterial phospholipid, which is applied in the field of molecular biology and achieves the effect of wide application prospect

Inactive Publication Date: 2015-05-27
JIANGNAN UNIV
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Problems solved by technology

At present, there is no information on the use of microbial source phospholipase A at home and abroad 2 The Report of Recombinant Expression of Genes in Kluyveromyces lactis

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  • Recombinant expression and application of lactobacillus casei phospholipase A2 gene
  • Recombinant expression and application of lactobacillus casei phospholipase A2 gene
  • Recombinant expression and application of lactobacillus casei phospholipase A2 gene

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Embodiment Construction

[0034] The present invention will be specifically described below in conjunction with the accompanying drawings. recombinant phospholipase A 2 Purchased from Jiangnan University China University Industrial Microbiology Resource and Information Center, Kluyveromyces lactis was purchased from New England Biotechnology Co., Ltd.

[0035] Expression of Recombinant Phospholipase A Using Kluyveromyces lactis GG799 Expression System 2 .

[0036] (1) Lactobacillus casei phospholipase A 2 Prediction of gene signal peptides:

[0037] Lactobacillus casei phospholipase A was found on NCBI 2 Gene sequence and the corresponding encoded amino acid sequence, SignalP 4.1server (http: / / www.cbs.dtu.dk / services / SignalP / ) predicts the signal peptide of the amino acid sequence, and the prediction results are as follows figure 1 shown.

[0038] (2) Construction of recombinant expression vector pKLAC1-pla2:

[0039] According to the prediction results of the signal peptide, the genome of Lacto...

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Abstract

The invention relates to a recombinant expression method of a lactobacillus casei phospholipase A2 gene. The recombinant expression method of the lactobacillus casei phospholipase A2 gene sequentially comprises the following steps: predicting a signal peptide of the gene, constructing a recombinant carrier, converting, screening, detecting an expression product, producing enzyme by recombinant bacteria, and fermenting; concretely firstly predicting the signal peptide of the gene according to the sequence of the lactobacillus casei phospholipase A2 gene, introducing a restriction enzyme cutting site at the two ends of a mature peptide by utilizing a specific primer, subcloning the mature peptide into a carrier pKLAC1 to obtain an expression carrier pKLAC1-pla2; then screening a recombinant lactic acid kluwe yeast strain to realize expression of recombinant phospholipase A2; then collecting supernate obtained through fermentation, and primarily detecting enzyme activity by adopting an egg yolk plate; and finally carrying out shaking fermentation cultivation on yeast. The recombinant expression method of the lactobacillus casei phospholipase A2 gene has the advantages that an eukaryotic expression vector is constructed according to the cloned lactobacillus casei phospholipase A2 gene, the lactobacillus casei phospholipase A2 gene is expressed by utilizing a yeast expression system, and influence of different fermentation conditions on growth of recombinant strains and enzyme production is studied.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a phospholipase A derived from Lactobacillus casei 2 Gene recombinant expression and its application. Background technique [0002] Phospholipase A 2 , the English name is phospholipase A 2 , abbreviated as PLA 2 , it can catalyze the hydrolysis of the two acyl groups of phospholipid glycerol molecules to generate lyso-lecithin and free fatty acids, and lyso-lecithin has important applications in the fields of food, cosmetics and medicine, and Japan has used it as a natural food additive. Therefore, it is an important enzyme in industrial production. [0003] Phospholipase A 2 Widely present in various organisms. It was first extracted directly from biological tissues, but there were problems such as low extraction rate and complicated operation. Some mammalian phospholipase A 2 Genes have been tried to express recombinantly in host bacteria such as Escherichia ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N9/18
Inventor 张梁王辉李由然顾正华丁重阳石贵阳
Owner JIANGNAN UNIV
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