Purification and characterization of phosphatidylcholine-specific phospholipase C from Spinach.

Abstract

To investigate the influence of the phospholipid degrading enzymesfor the quality of spinach during blanching and frozen storage, phosphatidylcholine-specific phospholipase C (PC-PLC) was partially purified from spinach and charaterized. The extract of spinach was heated at 50℃ and fractionated with ammonium sulfate between 10% and 60% saturation. A series of ion exchange chromatographies and affinity chromatography such as Q-sepharose, ConA-sepharose and SPsepharose chromatography, were applied to obtain the partially purified enzyme. Then the columns of Superdex 200 and Superose 12 connected in tandem were used for further purification. The apparent molecular weight of PC-PLC was determined to be about 13,000 Da by SDS-PAGE and MALDITOF MASS. The enzyme showed higher activity toward phosphatidylcholine than phosphatidylinositol and phosphatidylethanolamine. The optimum temperature and pH were 60℃, pH 7.0 respectively. The specific activity of PC-PLC was about 12mU/mg. A possible model was proposed in which the thermostable phosphatidylcholine-specific phospholipase C could influence the quality of spinach during the frozen storage.