Study On Enzymatic Synthesis Of Phosphatidylserine Enriched In N-3 PUFA

Phosphatidylserine (PS) is found widely exist in all cell membranes as a natural phospholipid. It’s especially abundant in animal brain tissue, occupied 10～20% of total brain phospholipids. PS plays an important role in brain function, such as recognition and memory, studies also show that PS has therapeutic effects on several memory-related disorders. This study examined the appropriate process for preparing PS from Antarctic krill phospholipids with phospholipase D (PLD).To monitor the reaction process, analysis methods for phospholipids were established firstly. Three methods, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and 31P nuclear magnetic resonance (31P NMR) were established for phospholipids analysis. Chloroform/methanol/water was used as the developing solvent in TLC method, the plate was colored with iodine vapor, and Gelpro32 software was combined used when phospholipids needed semi-quantitative analysis. In HPLC method, the analytical column was ZORBAX Rx-SIL, mobile phase was composed of n-hexane, isopropanol and water, ammonium formate was added to get better resolution, the detector was ELSD. All phospholipids could be separated by baseline after the elution program and ELSD parameter were optimized. In 31P NMR method, sodium cholate detergent was chosen to dissolve phospholipids, temperature and pH were optimized, and then all phospholipids except for SM and PE could be separated by baseline. All the three analytical methods have advantages and disadvantages, which one to choose in different parts of next studies based on different conditions.Then the enzymatic synthesis of phosphatidylserine enriched in n-3 poly unsaturated fatty acid (PUFA) from squid egg lecithin and L-serine was examined with the transphosphatidylation of PLD. Experiments were conducted to determine the optimal conditions. Firstly, they found that the synthesis of PS was significantly affected by the reaction system and the choice of PLD. After these two conditions were confirmed, single factor and orthogonal experiments were carried out to get the optimal process conditions for preparing PS. A biphasic system with ethyl acetate and 20 mM acetate buffer (pH 4.5), ethyl acetate volume to acetate buffer volume ratio of 5:6, reaction temperature of 40 ℃, PLD amount of 40 U/mL, squid egg lecithin to L-serine ratio of 1:32, reaction time of 6 h were used. PS content in the recovered phospholipid fraction increased to 30.90% under the optimal reaction conditions with the Antarctic krill oil substrate of 30.09% PC and 4.83% PE.The substrate specificity PLD catalyzed synthesis of PS was examined in the study. PC and phosphatidylethanolamine (PE) were separated and purified from five different kinds of sources including egg yolk, soybean, Antarctic krill, squid egg and sea cucumber. Then they were catalyzed to PS by the transphosphatidylation of PLD in the biphasic system. By comparing conversion ratio and reaction velocity of different reactions, results were obtained as follows:(1) conversion ratio of phospholipids originated from sea cucumber was lower than the other four origins because of the existence of ether-phospholipids, which implied that the existence of ether bond would suppress PLD activity; (2) less PE than PC was converted, which suggested that PC was easier to be converted with PLD than PE; (3) by correlation analysis of the degree of unsaturation of fatty acids of PC and kinetics of transphosphatidylation, results showed that reaction rate was higher with high poly unsaturated fatty acid (PUFA) content and low saturated fatty acid (SFA) content, substrate specificity of PLD to the five kind of PC was chicken egg> Antarctic krill, squid egg> soybean, sea cucumber. Studies showed that polar head group, fatty acids composition and bond connection could affect the transphosphatidylation of PLD on the synthesis of PS.A biphasic system of ethyl acetate and acetate buffer was used in this study to produce n-3 PUFA-PS, it was an alternative method for PS production. A new kind of functional factor was provided to study the brain function of n-3 PUFA-PS. Also it provided raw materials for the development of related functional products.