High Yield Of Phospholipase D Strain Screening,Identification And Its Activity Study

Phospholipase D is a kind of enzyme that hydrolyzes phospholipids to generate phosphatidic acid and a hydroxy compound, its catalytic site is phosphodiester bond of phospholipids. In addition to the hydrolytic activity, Phospholipase D also catalyzes the interconversion of polar head group of phospholipids by transphosphatidylation. Transphosphatidylation is particularly useful, it catalyzes abundant phosphatidylcholine to synthesis the natural rare phospholipids, such as Phosphatidylethanolamine(PE), phosphatidylserine(PS), phosphatidyl glycerol(PG). Phospholipase D derived from microorganisms has a strong ability to transfer phospholipids and broader substrate specificity; Among them, Phospholipase D that derived from the genus Streptomyces has the highest phosphoryl transfer activity.Phosphatidylserine is a membrane active substance. It plays an important role in improving memory, relieving stress of teenagers, preventing senile dementia, maintaining a healthy mood and so on. In nature, natural phosphatidylserine is very scarce, its extraction difficulty and high cost. Therefore, the preparation of cheap phosphatidylserine become the current hot spots. Compared with the extraction method, enzymatic preparation of phosphatidylserine has many advantages, such as mild reaction conditions, high purification rate, safety quality and others. Because of its unique physical and chemical properties and nutritional value, phosphatidylserine plays an important role in foods, cosmetics, medicines, and it has received international recognition.In this paper, a high yield Phospholipase D strain was selected from rapeseed oil soil by using the screening, TLC chromatography and HPLC quantitative analysis. The strain was identified as Streptomyces by morphology observation, physiological and biochemical identification and analysis of 16 s r RNA sequence, it is the highest homology with Streptomyces prunicolor. Against the strain fermentation culture conditions, we carried on the single factor experiments, the Plackett – Burman experiments and the response surface method(RSM) analysis. Finally the best enzyme production condition was determined: soluble starch 15.0g/L, the beef extract + peptone(1:1)15.0 g/L, K2HPO4 2.0 g/L, Mg SO4·7H2O 0.5 g/L, the initial p H 7.3, fermentation temperature 30.5℃, fermentation period 99 h. Under this condition, the enzyme activity reaches 36.06 U/L, its activity increased 65.3%, compared with the original fermentation medium.Under the optimum fermentation condition, enzymology properties of phospholipase D and its reaction conditions were optimized. Because of different enzymes, their nature are different; Even if they belong to the same kind of enzyme, but different sources can also cause the differences in enzymology properties. So the phospholipase D produced by this strain was carried out a series of studies: the optimum reaction temperature, the optimum p H, thermal stability, p H stability, preserve the time. Through a series of experimental analysis, its enzymology properties as follows: the optimal temperature 30℃, the optimal p H5.5; Temperature at 25℃-55℃, p H 4.0-7.5, preservation time within 30 d, the enzyme shows good stability. After studying the properties of the enzyme, in order to get higher conversion rate, a series of reaction conditions were optimized: reaction temperature, initial p H, concentration of Ser, different organic solvents, two phase volume ratio, different metal ions and reaction time. its optimum reaction conditions were obtained by single factor experimental analysis: 35℃, p H5.5, organic solvents as ethyl ether, Ether phase: water phase 2:1(V: V), metal ions as Ca2+ and under this condition reacted 4h. Above the conditions, the conversion rate of phosphatidylserine was 58.76%.