Heterologous Expression,Purification And Characterization Of Phospholipase C From Bacillus Cereus

Phospholipase C(phospholipase C,PLC,EC3.1.4.3)is a kind of hydrolase that catalyze the hydrolysis phospholipids to diacylglycerol and phosphocholine,phosphoinositide,phosphoric ethanol amine and so on.With the development of industrial requirements and the research of phospholipase C,phospholipase C application values have been gradually extended from the pharmaceutical production to oil refining,food processing,phospholipid modification and other fields.At present,domestic and abroad research of phospholipase C and its expressing host that face security risks in the field of food applications.In this study,phospholipase C from Bacillus cereus(B.cereus)was selected as the object of study,and Escherichia coli BL21/DE3(E.coli),Kluyveromyces lactis GG799(K.lactis)and Bacillus subtilis WB600(B.subtilis)were selected as expressing host.The recombinant expression vectors pET28abcplc1,pKLAC1bcplc2 and pHY300bcplc3 were transformed into Escherichia coli,Kluyveromyces lactis and Bacillus subtilis respectively.We successfully got the following recombinant strains: DE3/pET28abcplc1,K.lactis/pKLAC1bcplc2 and B.subtilis/pHY300bcplc3.The recombinant enzyme was purified to study its enzymatic properties.(1)DE3/PLC showed a target band near 30 kDa in SDS-PAGE.The optimum reaction temperature was 60? and the optimum pH was 9.0,and the enzyme activity was 15863U·mg-1 by p-NPPC method.DE3/PLC was stable at less than 45°C,pH 8.0-8.5.Mn2+,Mg2+ and Zn2+ significantly stimulated the enzyme activity of DE3/PLC.Whereas Cu2+ and Co2+ inhibited the enzyme activity,and Ca2+ had no obviously effect on the activity of DE3/PLC,the denaturation temperature of DE3/PLC was 65.3?.(2)GG799/PLC showed a target band between 35 and 40 kDa in SDS-PAGE.The enzyme activity was 19251 U/mg by p-NPPC method.The optimum reaction temperature was 80°C and the optimum pH was 9.0.GG799/PLC is more stable at less than 40°C,pH 7.0-8.0.Mn2+,Mg2+,Zn2+ and Ca2+ significantly stimulated the activity of GG799/PLC.Whereas Cu2+ and Co2+ inhibited the activity of GG799/PLC,the denaturation temperature of DE3/PLC was 81.6?.(3)WB600/PLC showed a target band near 30 kDa in SDS-PAGE.The optimum reaction temperature was 60? and the optimum pH was 9.0,and the enzyme activity was 15863U·mg-1 by p-NPPC method.WB600/PLC was stable at less than 45?,pH 8.0-8.5.Mn2+,Mg2+ and Zn2+ significantly stimulated the enzyme activity of DE3/PLC.Whereas Cu2+ and Co2+ inhibited the enzyme activity,and Ca2+ had no obviously effect on the activity of WB600/PLC,the denaturation temperature of DE3/PLC was 67.5?.(4)In this study,we optimized the purification method of phospholipase C: by removing the high concentration of imidazole through Desalting affinity chromatography.The DE3/PLC and WB600/PLC can be detected its activity after purification,which means we solved the problem of phospholipase C lost its activity after purification.Through the analysis of glycosylation sites of GG799/PLC and the identification of glycosylation,it was proved that phospholipase C expressed by Kluyveromyces lactis was modified by glycosylation.