Purification of plasmalogen and synthesis of phospholipid by phospholipases

Abstract

In order to purify plasmalogen of phospholipids from bovine heart and brain, hospholipase A1 from Serratia sp. MK1 was used. This enzyme hydrolyzes only the ester bond at sn-1 position of diacyl glycerophosphocholine, not the ether bond of plasmalogen. We, therefore, obtained higher purity of plasmalogen of phospholipids from bovine heart and brain with the phospholipase. For the selection of the suitable reaction system, the reaction was examined in a two-phase system and an emulsion system. The purity of plasmalogen in the two-phase system was found to be better than that obtained in the emulsion system. In the case of the emulsion system $CaCl_2$ was required and 60% of diacyl glycerophosphocholine was hydrolyzed. However, in the two-phase system no $CaCl_2$ was required and most of diacyl glycerophosphocholine was hydrolyzed within 45 min. It was found that Tris-Cl buffer was better than phosphate buffer for the reaction. In Among various solvents, butyl acetate showed best catalytic activity and enzyme stability. Therefore we selected butyl acetate as a solvent in a two-phase system. The different ratios of solvent-aqueous phase had no significant effects on the purity of plasmalogen. In case of ethanolamine containing substrate, diacyl glycerophosphoethanolamine was completely hydrolyzed. When crude plasmalogen containing phospholipid was used, 99% purified plasmalogen was obtained at 0.5% substrate concentration. Synthesis of useful phospholipid with phospholipase $A_2$ was investigated. We synthesized phospholipids with PUFA (poly unsaturated fatty acid) and selected EPA (eicosapentaenoic acid) as a PUFA. For the increasing yield of PUFA-phospholipid, optimal EPA and enzyme quantity was investigated. Optimal EPA (using EPA-ester as a acyl donor) quantity was 50 mg when LPC was 10 mg. Phospholipase quantity was determined to 5 mg in considering reactant viscosity and enzyme cost. There are various parameters that govern this PUFA-phospholipid synthesis ...