Cloning, Expression And Characterization Of Phospholipase C Gene

Phospholipase C (PLC, EC3.1.4.3) is a kind of enzymes that hydrolysis phospholipids todiacylglycerol (DAG) and the original phosphorylated head group. PLC is wildly used indegumming of oil, processing of foods and pharmaceutical science.PLC playing a significant role in bacterial pathogenesis is found in bacteria. PLC fromAcinetobacter calcoaceticus (A. calcoaceticus), Streptomyces venezuelae (S. venezuelae) andBacillus cereus (B. cereus) were researched in this study. The PLC activity was determined byp-NPPC methods. The PLC substrate specificity was determined by inorganic phosphorusmethods and TLC. In the paper, four PLC were ligated with pET28a(+), and expressed in E.coli. Recombinant PLCs were purified using affinity chromatography.(1) The PLC activity in culture supernatant of A. calcoaceticus, S. venezuelae and B. cereuswere comfirmed by p-NPPC method and egg yolk method. PLC gene sequences weredownload from NCBI database. Gene acplc1and acplc2were from A. calcoaceticus. Genesvplc1, svplc2and svplc3were from S. venezuelae. Gene bcplc was from B. cereus.(2) PLC in A. calcoaceticus, S. venezuelae and B. cereus were amplified by using PCR. Weinserted plc into pET28a(+) and transformed into E. coli BL21(DE3). PLC was purified withaffinity chromatography to analyze enzymatic properties. Cell lysate supernatant ofpET28a-acplc1/BL21show a88kDa target band in SDS-PAGE. Cell lysate supernatant ofpET28a-acplc2/BL21show a85kDa target band in SDS-PAGE. The enzyme activities ofacPLC1and acPLC2were (31160±418) U·mg-1and (13640±354) U·mg-1; svplc1, svplc2,svplc3from S. venezuelae were also expressed in E. coli. The cell lysate supernatant ofpET28a-svplc3/BL21showed significantly p-NPPC activity, whereas pET28a-svplc1/BL21,pET28a-svplc2/BL21did not show p-NPPC activity or egg yolk activity. Cell lysatesupernatant of pET28a-svplc3/BL21show a79kDa target band in SDS-PAGE, and theenzyme activity was (7404±101) U·mg-1with p-NPPC method. Cell lysate supernatant ofpET28a-bcplc/BL21show a33kDa target band in SDS-PAGE. bcPLC showed both p-NPPCactivity and egg yolk activity.(3) The analysis of enzymatic properties showed that the optimum temperature and pH were65°C,8for acPLC1, and50°C,7.5for acPLC2. acPLC2was stable at30°C, pH7. Mg2+andCa2+stimulated the activities of acPLC1and acPLC2, whereas Zn2+stimulated acPLC1andinhibited acPLC2. acPLC1performed poorly on phospholipids. acPLC2hydrolyzedphosphatidylinositol and performed poorly on the other substrates. The analysis of purifiedsvPLC3showed the optimum temperature and pH were60°C,9. svPLC3was stable at45°C,pH8.5-9. Mg2+and Zn2+stimulated the activities of svPLC3, whereas Ca2+and Ni2+inhibitedsvPLC3. svPLC3performed poorly on phospholipids. The analysis of bcPLC propertiesshowed that the optimum temperature and pH were60°C,7.5, bcPLC was stable at45°C, pH8-9. Mg2+, Zn2+and Ni2+stimulated the activities of bcPLC, whereas Cu2+inhibited bcPLC.The analysis of substrate specificity showed that bcPLC could hydrolyze phosphatidylcholin,phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. In view of bcPLChydrolysis of various phospholipids and thermal stability advantages, bcPLC has a good application prospect in vegetable oil degumming.