| Phosphatidylserine plays an important role in improving the memory, treating ADHDand depression, and preventing Alzheimer’s disease. However, the content of ps is rare innature, so the preparation of phosphatidylserine with a good quality and high purity is verynecessary. Enzymatic preparation of phosphatidylserine means the reaction of serine andphospholipids catalyzed by phospholipase D under certain conditions. Compared to the others,enzymatic preparation has many advantages, which can be carried out under mild conditionswith less by-products, high product yield, and good quality products. In the work, thefermentation conditions for preparing phospholipase D with Streptomyces sp.CA-1werestudied, besides, the purification and characterization of phospholipase D, the basic process ofenzymatic preparation of phosphatidylserine in biphasic reaction system were also studied.The optimum fermentation conditions for enzyme production with Streptomycessp.CA-1were obtained by single factor and orthogonal experiments：glucose20g/L, beefextract8.75g/L+fish peptone8.75g/L, Tween2032.5g/L, calcium chloride2.5g/L,K2HPO42g/L，MgSO4·7H2O0.5g/L,1.5%of inoculums (v:v), medium initial pH7.0, culturetemperature30℃, culture time3d. Under the optimum conditions, an enzyme ctivity of1605.9U/L was reached, which is7.72times higher than the enzyme ctivity obtained beforeoptimization. The factor influence on the activity showed by orthogonal experiment was listedin a decreasing order as follows: complex nitrogen source> Tween20> glucose> calcium,furthermore nitrogen and tween20were found to be the most important on the preparation ofphospholipase D.The crude enzyme solution was purified by centrifuging, filtering, salting out, dialysisand concentrating, gel filtration, and ion-exchange chromatography. After purification, thehydrolytic activity of phospholipase D was116.32U/mg, with a recovery rate of19.73%,which is purified14.71times. The molecular weight of purified PLD was estimated to beabout53kDa by SDS-PAGE.The optimum temperature and pH of hydrolysis reaction catalyzed by phospholipase Dwere showed to be35℃and4.5, when the temperature is below55℃, pH of4.0-7.0, theactivity showed a good stability. The optimum temperature and pH of phospholipase D forcatalyzing transesterification were40℃and4.5, when it is below50℃and a pH of4.0to7.0,the activity has good stability. The hydrolytic and transesterification activity can be promotedby calcium and magnesium ions. Furthermore, with the action of calcium ion the hydrolysisactivity was increased by6.1%and transesterification activity was increased by13.7%, whileit is strongly inhibited by manganese ion with hydrolysis activity decreased by47.1%andtransesterification activity decreased by58.4%. The enzyme structure was measured by themethod of CD, the results showed that with the addition of calcium ion or magnesium ion, thestructure of enzyme became more stable with the ordered structure increased and thedisordered structure reduced. Enzyme was showed an unfavorable stability with the decreaseof α helix and β sheet content with the addition of manganese ion, and the activity decreased. Enzymatic catalytic reactions were conducted in a biphasic reaction system, with thesubstrates of phosphatidylcholine and L-serine. The basic process conditions for enzymaticpreparation of phosphatidylserine were studied. Results showed that the optimal buffer of0.2mol/L acetic acid-sodium acetate, ethyl acetate as the organic phase, the phosphatidylcholineconcentration of10mg/mL, the ratio of phosphatidylcholine to L-serine1:3, volume ratio oforganic phase to aqueous phase2:1, the reaction temperature of40℃, pH4.5, enzyme amount8mg, calcium chloride15mmol/L, and under this condition, phosphatidylserine of67.8%was attained. |
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