Screening And Optimization Of Phospholipase C Strain, And Its Heterologous Expression

Phospholipase C,(PLC,EC3.1.4.3), is a kind of hydrolase that catalyzes the hydrolysis ofphospholipids to yield diacylglycerol and a phosphate monoester, such as phosphorylcholine,phosphoinositide, phosphoric acid ethanol amine and so on. With the development of researchon PLC and the needs of the industry development, PLC application values have beengradually explored. It has been widely used in the oil refining, food processing, phospholipidsmodification and pharmaceutical production. A PLC producing strain Bacillus cereus12wasisolated and its PLC gene was cloned and expressed in E.coli and Pichia pastoris. Specificresearches are as follows:PLC producing stain Bacillus cereus12was isolated from600strains of bacteria on theegg-yolk solid medium. By single-factor experiments the optimal conditions for Bacilluscereus12fermenting were obtained to be2%inoculation amount,200r/min at32℃,0.2mmol/L Zn2+and0.75%(v/v) egg yolk. Under these conditions the PLC activity was up to23.78±0.32U/mL which had been enhanced by26.50%.The PLC genes pcplc1(with peptide) and pcplc2(without peptide) of Bacillus cereus12were successfully obtained by PCR. Two recombinant plasmids were constructed andtransformed into Escherichia coli BL21(DE3). Induced by IPTG (isopropylβ-D-1-thiogalactopyranoside), an approximate33kDa protein with significant PLC activitywas detected in recombinant with pcplc1by SDS-PAGE. Under the optimal inductioncondition:0.2mmol/L IPTG added at1.5th hour and induced for4hours under25℃,maximum PLC activity (30.24±0.18U/mL) was obtained.Properties of the active recombinant enzyme were observed. The results showed that itsoptimal reaction temperature was80℃and optimal reaction pH was6.8. The activity of therecombinant PLC was stable under40℃for30min but decreased sharply under60℃. It wasstable in reaction buffer of pH6.0-7.6for1h but reduced greatly below4.0or above9.0. Theactivity of recombinant PLC was inhibited by Cu2+obviously and was increased by Zn2+andMn2+. Ni2+was not so sensitive to its activity. By increasing the concentration of Na+andMg2+, the inhibition on PLC activity was getting stronger. The recombinant enzyme in E.colipurified by His Trap Chelating column was detected as electrophoretically pure bySDS-PAGE.Recombinant plasmids pPIC9K-pcplc1and pPIC9K-pcplc2were constructed andtransformed into Pichia pastoris GS115and X-33by linearizing with SacI. Recombinantsscreened by MD plates were used to aquire multicopies with different concentration of G418.Several recombinants tested to be Mut+by PCR were induced by0.5%methanol at30℃andPLC acticity was successfully detected on borax-egg yolk plate.