The Research On The Reaction Of Phospholipase D Catalyzed Transphosphatidylation For The Syntheses Of Phosphatidylserine

As an important biological membrance phospholipid material, Phosphatidylserine(PS), which is an active substance, can regulate protein on the cell membrance. It has significant physiological function, such as improving the function of nerve cells and the brain memory, adjusting the transmission of nerve impulses, etc. Therefore, it has a very wide of uses in health care industry and pharmaceutical industry. However, the natural PS are very rare, which is not enough to satisfy mans’ demand for PS, thus it is very necessary for the synthesis of phosphatidylserine. The enzymatic conversion method is a common synthesis method of PS with mild conditions and low production costs. In this paper, research on the reaction rule of phospholipase D catalyzed phosphatidylcholine (PC) transphosphatidylation for the syntheses of phosphatidylserine, which provide theoretical reference for the industrial application.PLD-catalyzed transphosphatidylation for the syntheses of phosphatidylserine was carried out in biphasic reaction system in which PC was dissolved in the organic solvent phase, while L-serine was dissolved in buffer phase. The effects of organic solvent, substrate malar ratio, pH, two phase volume ratio and reaction temperature were investigated. The optimal reaction condition of preparation of phosphatidylserine is as follow:organic solvent is ether, the reaction temperature is 28℃, the substrate malar ratio is 100, pH value is 5.5, two phase volume ratio is 2. Under the conditions, the concentration of PS in reaction liquid is 7.57mmol/L, the yield of PS is 58.4%.Studied the effect of phospholipase D catalyzed transphosphatidylation by-products choline, then propose the kinetics model of enzymatic reaction and establish the relevant kinetic equation. By simulationing catalytic reaction rate of several different concentrations of choline, and using the L-B method for data, we confirme that choline is a competitive inhibition for phospholipase D catalyzed reaction and obtain the kinetic parameters, under 28℃ and pH=5.5, Michaelis constant and inhibition constant were 15.7mmol/L å'Œ 9.16 mmol/L, respectively.Free enzyme is sensitive to the environment, easy to inactivation and hard to separation from the reaction. Adopting immobilized method to integrate the enzyme and carrier, it is not only to separate easily, but can be reused. The results show that thermal stability of immobilized enzyme has been increased. The residual enzyme activity was 40% after immobilized enzyme was repeated use for 5 times, more than 32% immobilized enzyme residual activity is maintained after 50 days of storage in 4℃, while free enzyme has been deactivation. In order to remove choline, inhibiting the progress of the reaction, we adopt the method of batch charging. The repeated batch operation method are adopted to decrease the choline in the reaction system, increase the catalytic reaction rate and product yield, the yield of PS is 58.9%.