Cloning And Expression Of Phospholipase A₂Gene From Lactobacillus Casei

The mature protein gene pla2a was cloned from Lactobacillus casei by PCR. The recombinant plasmid pMD-pla2a was constructed and the results of homologous comparison were up to99%. The followed expression plasmid pET28a-pla2a was successfully constructed and transformed into E. coli DE3. As well as, the activity of PLA2expressed successfully in all of the recombinant bacteria was detected on borax yolk tablet. The molecular weight was determined as17kDa by SDS-PAGE. The fermentation conditions of E. coli (pET28a-pla2a) was researched by measuring the activity of the enzyme. The optional conditions cultivated in LB medium and induced by IPTG was that add time was2h, induction temperature was25℃, IPTG concentration was0.1mmol/L and induction time4h and the maximum activity was2.8±0.2U/ml. Then the optional condition induced by lactose was that induction opportunity was2h, temperature was25℃, induction time was6h and the maximum activity was2.5±0.2U/mL. In TB medium, the condition induced by lactose was that add time was3h, temperature was25℃, induction time was10h and the maximum activity was3.0±0.2U/mLThe complete structure gene pla2b was cloned from L. casei by PCR. Expression plasmid pHY-P43-pla2b and pPIC-CP-pla2a were also successfully constructed and transformed into Bacillus subtilis WB600、Pichiapastoris GS115separately The maximum activity of recombinant B. subtilis WB600(pHY-P43-pla2b) and P. pastoris GS115(pPIC-CP-pla2a) was detected. The results showed that the activity of recombinant B. subtiliswas1.5±0.2U/mL and the activity of P. pastoris was2.0±0.2U/mL.The purity of the protein purified by Ni-chelating-column can be up to95%, and then the analysis of the enzyme properties was followed. The results showed that the optimal temperature was37℃and the optimal pH was8.0. In additional, the activity of the recombinant phospholipase A2was more stable during the temperature below50℃, as well as, the activity was invariant at65℃for1hour.The recombinant phospholipase A2was the hydrolysis and the soybean phospholipid was the substrate in this experiment. The optimal condition was that concentration of enzyme was2.5U/g, reaction temperature was37℃, initiative reaction pH8.0, concentration of Ca2+was50mmol/L, and reaction time was8h. The quantity of Fat acid was781μmol and the average convert ratio was60.6%.