Heterologous Expression Of Phospholipase A1Gene From Citrobacter Youngae

Phospholipase A1(PLA1, EC3.1.1.32) is an enzyme that hydrolyzes phospholipids andproduces2-acyl-lysophospholipids and free fatty acids. Traditional PLA1was mainlyobtained from the venom and animal pancreas. This method has a lot of problems in itspurification and production, so it can not meet the needs of industrial production. Microbialenzymes possess strong appication values because of their great advantages, such as simpleoperation, short production period and easy to large-scale production. But both themicroorganisms producing PLA1and the researches on heterologous expression of PLA1arefew, the enzyme activity is generally low. So, this experiment is mainly intend to study theheterologous expression of PLA1from microorganisms. And the detailed contents and resultsare as follows:(1) The measure methods of PLA1activity were studied by comparative analysis of yolkplate method, alkali titration and spectrophotometric. Consequently, yolk plate method waschosen for the qualitative and semi-quantitative of PLA1activity while the alkali titration forquantitative analysis.(2) The PLA1gene pla1from C. youngae CICC No.21596was obtained by PCR. Therecombinant E.coli was successfully constructed. After induction of IPTG, an approximately33kDa recombinant protein with obvious PLA1activity was detected by SDS-PAGE andyolk plate method. Under the optimal induced condition:0.4mmol·L-1IPTG added whenOD600come to0.6and induced8h at temperature of37℃, a maximum PLA1activity(5.6±0.2U·mL-1) was obtained.(3) The recombinant enzyme was purified by Ni-chelating column. Then the enzymaticproperty was studied. The results showed that the recombinant enzyme performed themaximal activity at50℃, pH7.5. And this enzyme had thermostability between20℃and60℃and pH stability from pH6.0to8.0.(4) The PLA1gene pla1from C. youngae was cloned by PCR. The expression plasmidpPIC9K-pla1and pHY-p43-pla1were successfully constructed and transformed into Pichiapastoris GS115and Bacillus subtilis WB600separately. The activities of PLA1produce bytwo recombinant strains were compared. The results showed that PLA1activity ofGS115/pPIC9K-pla1was significantly higher than that of WB600/pHY-p43-pla1.(5) The fermentation conditions of the recombinant P.pastoris GS115strain werestudied. The optimizing culture conditions were as follows: inoculum70mL/50mLfermentation medium, added0.4%methanol, adjust pH6.0, induction time102h.Under the condition, the recombinant enzyme activity of fermentation supernatant was3.73U·mL-1.