Preparation of highly active phospholipase d and yeast whole cell catalyst displaying phospholipase d on the cell surface

Abstract

The invention relates to preparation of high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts, which belongs to a method for carrying out site directed mutagenesis wild phospholipids enzyme D by recombinant deoxyribonucleic acid (DNA) for improving the activity, connecting the mutated gene with yeast show carriers pPIC9K-Flo and efficiently displaying the mutated gene on the surface of pichia pastoris cells, and relates to a preparation method of the high-activity phospholipids enzyme D and cell surface display phospholipids enzyme D yeast whole cell catalysts. The method has the advantages that the activity of the wild phospholipids enzyme D is improved and is shown on the surfaces of the pastoris cells, the stability is improved,and the advantages of immobilized enzymes are realized. The method has the technical scheme that wild phospholipids enzyme D genes are separated from microbes, particularly streptomyces aureofuscus, the mutation is carried out on the amino acid residue of Glu69 and Ser285, through the efficient expression on the surfaces of the pichia pastoris cells, the enzyme activity of the high-activity phospholipids enzyme is improved by 11 percent than the wild type phospholipids enzyme D, and the enzyme activity of the recombination strains GS115 / pPIC9K-Flo-pldm prepared through high-density fermentation is 120U / (g. stem cells).

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to site-directed mutation and recombination of genes, in particular to the preparation of a high-activity phospholipase D and a yeast whole-cell catalyst displaying phospholipase D on the cell surface. Background technique [0002] Phosphatidylserine (PS) exists in the cell membranes of all animals, higher plants and microorganisms, and is one of the phospholipid components of cell membranes. PS content is low in animals, plants and microorganisms, accounting for only 2-10% of the total phospholipids. PS in the human body is mainly concentrated in the brain, accounting for about 15% of the total amount of phospholipids in the brain. PS can only be synthesized in a small amount in the human body, mainly by intake from food. PS regulates the activity of its receptors, enzymes, ion channels, messenger molecules, etc. by directly acting on proteins in the membrane and membrane-associated p...

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Problems solved by technology

[0004] At present, the mainstream method of industrial production of PS is the organic solvent extraction method, which has many shortcomings: 1. This method consumes a large amount of organic solvents, which is harmful to the environment; The properties are similar, and the content of PS is relatively small. Therefore, the extracted PS has low purity and often contains other phospholipids, such as lecithin, phosphatidylinositol, phosphatidylethanolamine, etc.; 3. The last and most critical issue is The content of PS in the raw material is low, even if the soybean that is rich in PS is used as the raw material, this problem also exists. The phospholipids in the soybean account for 1.6-2% of the total weight of the soybean, and PS only accounts for 0.5-1%, that is, Each kilogram of soybeans only contains 80-200mgPS, plus the cost of separation and purification, so the price of the product continues to rise

[0009] Free phospholipase D has poor stability and cannot be recycled. Although the enzyme immobilization technology can solve the above problems, the traditional immobilization method will also produce some disadvantages. For example, the immobilization process may cause the loss of enzyme activity and yield; in addition , because the immobilization operation requires a carrier, thus increasing the cost of the carrier and the cost of the immobilization operation, and the yeast cell surface display technology provides a new biological method based on gene recombination technology for the immobilization of enzymes

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