Preliminary Studies On Preparation And Catalytic Reaction Of Phospholipase D

Phospholipid is widely used in food, pharmaceutical industries and other fields, under most circumstances, natural phospholipids can not meet the requirements of the preformance. Phospholipase D is the key technology used successfully in preparing semi-synthetic phospholipids, so the enzyme technology was concerned and developed. In this text, the characterization of phospholipase D secreted by actinomyces and application of transphosphatidylation was preliminary studied.Phospholipase D secreted by actinomyces was preliminary purified by (NH₄)₂SO₄ precipitation, ethanol precipitation, PEG precipitation respectively. By means of (NH₄)₂SO₄ precipitation(in 60% saturation), the phospholipase D was purified 8.01 times, and the recovery ratio was 50.9%; By means of ethanol precipitation(in the volume ratio 1.0:1 between ethanol and raw enzyme), it was 8.08 times and the recovery ratio was 60%; By means of PEG precipitation (in 0.4g/ml concentration), the phospholipase D was purified 11.2 times, and the recovery ratio was 50.9%.The hydrolysis activity of phospholipase D was determined by a spectrophotometric assay under different conditions. the result showed the phospholipase D has maximal hydrolysis activity at pH 6, 50°C; phospholipase D was active and highly stable in solution from pH 4 to 8 ; its activity was steady when the temperature bellowed 40°C. Phospholipase D had a better stability in preservation, the enzyme activity would keep above 80% below 4°C for 30 days.The immobilization of the phospholipase D with diatomite, kaolin, silica gel as the carrier was studied. By comparison of immobilization results of three carrier, it was found that diatomite is the optimal carrier.A new non-ionic surfactant called glutamic acid didodecyl ester ribitol amide was synthesized from L-glutamic acid, lauryl alcohol, glucone-δ-lactone, and was used to coated phospholipase D. When the ratio between the surfactant mass and jprotein mass of the phospholipase D was 2:1, we could get the maximal coated rate of 40%.The preliminary research was made on the reaction of phosphatidylcholine(PC) catalyzed by PLD in organic solvent, and the results showed when the concentration of alcohol receptor far exceeding PC, the reaction of transphosphatidylation and hydrolysis catalyzed by phospholipase D would coincident with Michaelis-Menten kinetic equation. The result of transphosphatidylation of PC catalyzed by the native PLD, immobilized PLD, lipid-coated PLD showed that the transphosphatidylation activity of a lipid-coated PLD was 20 times than the native PLD.The research had important use for reference on synthesizing and semi-synthesizing phospholipid from Phospholipase D and natural phospholipids.