Preparation Of Lysophosphatidylethanolamine Catalyzed By Phosphpolipase A₁ In Aqueous Medium And Acyl Migration Mechanism

Lysophosphatidylethanolamine is a hydrolytic material by phospholipase A1-catalyzed a molecule fatty acid of phosphatidylethanolamine. Lysophosphatidylethanolamine, for having important medical applications, such as anti-HIV and anti-tumor, liposomes as drug carriers, is widely used in medicine industry. Specifically, researchers are becoming increasingly about the knowledge of LPE medicinal function. Therefore, the development and application of high-purity Lysophosphatidylethanolamine is also highly anticipated. Unfortunately, LPE is scarce in natural sources, no high-purity Lysophosphatidylethanolamine. The current work described a simple method for producing LPE by the green enzymatic technology and the silica gel column chromatography technology, which solved the problem of the low purity, low conversion rate, and environmental protection. And meantime, the auto-acyl migration mechanism was also studied.Firstly, the technology of environmental friendliness enzymatic preparation of the Lysophosphatidylethanolamine was established in this study. Lysophosphatidylethanolamine was prepared by phospholipase A1 hydrolysis of phosphatidylethanolamine in an aqueous system. The effects of the ratio of liquor to material, temperature, enzyme loading, and dosage of Ca Cl2 on the relative content of LPE were discussed. Experiments were designed by orthogonal experiment for optimization of the effects of the single experimental factors on the relative content of LPE. The optimal condition was confirmed as follows: the purity of PE 80%, the ratio of liquorto material 10:1, temperature 35 ℃, dosage of phospholipase A1 0.04 m L/g, dosage of Ca Cl2 0.01 g/m L, the relative content of LPE reached 42.0%.The silica gel column chromatography to purify the Lysophosphatidylethanolamine was established. The factors including ratio of elution solution, the sampling amount, the sample concentration, the flow rate were studied. Under these conditions, the optimization of the purification process were studied. The optimal experimental condition was confirmed as follows: the sampling amount 1:40 g/g, sample concentration 30 mg/m L, the flow rate 2.5 m L/min, the purity and recovery of Lysophosphatidylethanolamine was 99.05% and 88.69%.The acyl transfer mechanism of the Lysophosphatidylethanolamine was studied. Phospholipase A1 hydrolysis progress was detected by HPLC-ELSD. The result proved that acyl migration phenomenon existed in the process of enzymolysis. In the same way, phospholipase A1 and phospholipase A2 hydrolysis progress was detected by HPLC-RID respectively. Sn-1 fatty acid of PE was hydrolyzed by phospholipase A1 to prepare Sn-2-LPE, then spontaneous acyl migration occurred in which the Sn-2 acyl moved to form Sn-1-LPE.The effect of some single reaction factors, including the polarity of the liquor, p H, temperature and substrate concentration.