Heterologous Expression Of Streptomyces Phospholipase D In Pichia Pastoris

Phospholipase D(PLD)belongs to the phospholipase superfamily.It is an enzyme that acts on phosphoryl oxygen bond and catalyzes the hydrolysis of phospholipids into choline and phosphatidic acid.Under certain conditions,it can also catalyze the reaction of other hydroxy compounds with the bases of phospholipids to generate new phospholipids.Phospholipase D can be used to modify common phospholipids,which can produce rare phospholipids and improve biological value.Phospholipase D widely exists in various groups in nature,but the complex extraction process of phospholipase D from animal origin and plant origin and a large number of impurities in the product,the production of phospholipase D from wild-type microorganisms is extremely low,these factors limit the large-scale production of PLD and its downstream industry.With the development of biological science and technology,at present,people are more inclined to obtain the strains with high expression of enzyme preparation and the protein structure of phospholipase D by means of molecular biology and genetic engineering.According to previous studies,the Streptomyces PMF-PLD has good hydrolytic activity and transphospholipid activity to lecithin,it also has high tolerance to the substrate of enzymatic reaction,is currently widely used.In the studies on the heterogenic expression of phospholipase D,the E.coli expression system was the most used.However,in the process of protein expression,the inclusion body problem is prominent and phospholipase D has an inhibitory effect on the growth of E.coli,the expression is not good.In this paper,we studied the heterologous expression of Streptomyces PMF-PLD in Pichia pastoris protein expression system.The experiment mainly used the original recombinant plasmid of Streptomyces PMF-PLD preserved in laboratory,by means of gene cloning,vector construction,E.coli transformation and plasmid extraction,we obtained the expression vectors pPICZ?A-PLD and pET-28a-PLD as well as the corresponding Pichia pastoris strains X-33/pPICZ?A-PLD and E.coli BL21(DE3)/pET-28a-PLD.Then we explored the expression of phospholipase D.SDS-PAGE and Western blot showed that the phospholipase D of Streptomyces could be expressed in Pichia pastoris,proved that we have obtained the corresponding strains,but the expression level is not as good as expected.Compared with the prokaryotic expression system,the heterologous expression of phospholipase D in Pichia pastoris and other eukaryotic expression systems is still in the initial stage.In view of the unique advantages of Pichia pastoris in protein expression,the experiment will provide the possibility for the high level expression of phospholipase D.