Study On Cloning And Expression Of Phospholipase C From Listeria Monocytogenes And The Application In Oil Degumming

In order to develop a new PLC for the oil degumming, the phospholipase C gene(lm-plcB) from Listeria Monocytogenes was cloned and expressed in E.coli and thefermentation conditions were optimized. The enzymatic properties of PLC was investigatedand the recombinant PLC was used for the degumming of different crude oils. The maincontents of this study were as follows:The phospholipase C gene (lm-plcB) from Listeria Monocytogenes was cloned andsubsequently, recombinant E.coil BL21(DE3)/pET28a-lm-plcB was constructed and anapproximate30.4kDa recombinant protein was detected by SDS-PAGE. The results showedthat PLC was successfully expressed in E.coil BL21(DE3)/pET28a-lm-plcB. The analysesof yolk agar plate test showed that the recombinant protein had a significant PLC activity withthe natural phospholipid as the substrate.The optimal fermentation conditions for E.coil BL21(DE3)/pET28a-lm-plcB wereobtained with4%of inoculation amount,200r/min for2hours at the temperature of37℃,and induced by2.5g/L lactose for26hours at25℃. Under these conditions，the ultimateenzyme activity of PLC could reach754.6U/mL.When the PLC was purified byion-exchange chromatography (Q-Sepharose High Performance), the specific activity of PLCwas473.1U/mg.Properties of the recombinant enzyme PLC were studied. The optimal temperatureobserved was60℃. When the PLC was kept at60℃for30min, more than95%of its initialactivity was preserved. However, enzyme activity decreased significantly at the temperatureabove60℃. The optimum pH was observed at5.0, and the PLC was stable in a pH range of6.0to7.0.The activity was increased in presence of Zn2+, Ba2+, Mg2+, Mn2+, whereas Cd2+had negative effect on its activity. Ca2+, Fe3+had little effect on PLC activity. Moreover, thisPLC exhibited broad phospholipid substrate specificity towards PC, PE and PI, but showed nohydrolysis effect to PA.Finally, the recombinantional PLC was applied to oil degumming，and four differentkinds of plant crude oils were chosed. The phosphorus content of soybean crude oil (Highcontent of phospholipids) was reduced from398.22mg/kg to5.28mg/kg，and98.67%phosphorus was removed. The phosphorus content of soybean crude oil (Low content ofphospholipids) was reduced from216.67mg/kg to3.71mg/kg,with98.40%phosphorusremoval rate. The phosphorus content in rapeseed crude oil was decreased from183.71mg/kgto4.50mg/kg with97.55%phosphorus removal rate. The phosphorus content in rice brancrude oil was reduced from306.82mg/kg to50.19mg/kg with83.63%phosphorus removalrate. The optimum conditions of degumming test of the soybean crude oil high in phosphoruswere determined as follows:55℃of temperature, pH5.5,1000U/kg oil,1.5h of reactiontime. Under such conditions, the residual phosphorus content in degummed oil was decreasedto4.28mg/kg, which can totally meet the requirement of the physical refine, and alsoincreased oil yield by0.5%. In summary, the lm-plcB gene from L.Monocytogenes was cloned and expressed inE.coli BL21(DE3), and then the recombinantional phospholipase C was used in oildegumming process. The degummed oil treated by the recombinantional phospholipase C canmeet the requirement of physical refining, and the refining rate can also be increased slightly.So the recombinantional phospholipase C has a good prospect in the edible oil refiningindustry.