The Study On Expression Of Phospholine A₂ In Pichia Pastoris And Its Application In Synthesit Of 2-DHA-PS

Phosphatidylserine containing docosahexaenoic acid residue at the sn-2 position(2-DHA-PS)is a particularly important phospholipids,which show physiological functions of phosphatidylserine and docosahexaenoic acid.2-DHA-PS has been shown to be capable of ameliorating Alzhemier,improving cognitions and used to treating brain atrophy,depression and children with attention-deficit hyperactivity disorder.The preparation method of 2-DHA-PS could not produce high purity products.To overcome the limitions of the above processes,enzymatic method using phospholipase D(PLD)and porcine pancreatic phospholipase A2(PLA2)were explored for production of highly pure for 2-DHA-PS in this study.To achieve heterologous production of 2-DHA-PS,the synthetic gene plA2m was inserted into the plasmid pPIC9K and transformed into Pichia pastoris GS115,resulting in construction of recombinant GS115/pPIC9K-plA2m.The fermentation conditions of recombinant GS115/pPIC9K-plA2m were optimized in shake falsk at single factor levels.It was found that the optimum medium and fermentation conditions for GS115/pPIC9K-plA2m were:methanol 1.5%(v/v),initial pH 6.5,temperature 28?,volume 12.5ml,speed 300r/min.The PLA2 activity was up to 38.4 U/ml,an increase of 72.4%compared to that with the original conditions(22.4 U/ml).Studies of enzymatic properties of PLA2 showed that its optimum temperature was 40? and the optimum pH 8.0.PLA2 was stable at pH 6.0-9.0,while it showed good thermal stability at 30-40?.To evaluate the enzymatic preparation process of 2-DHA-PS,PS was synthesized by PLD with phosphatidylcholine and serine as substrates,followed by 2-DHA-PS production by PLA2 with Phosphatidylserine and docosahexaenoic acid as substrates,leading to the production of 2-DHA-PS with a conversion rate of 3.01%.