Expression Of Hemolytic Phospholipase C From Pseudomonas Aeruginosa In Escherichia Coli And Optimization Of Fermentation Conditions

Phospholipase C (PLC) is a kind of lipid hydrolase. A high-producing PLC strain ofPseudomonas aeruginosa was isolated, and its PLC genes were cloned and espressed inrecombinant Escherichia coli, and then optimized their fermentation conditions. Researchresults were as follows:PLC producing strain of Pseudomonas aeruginosa (P. aeruginosa)41was screenedthrough yolk borax plate method and NPPC method, and its PLC genes (plcH, plcN, plcR andplcB) were cloned. The recombinant expression plasmids were constructed and transformedinto Escherichia coli BL21(DE3), and determined the PLC activity or hemolytic activity. Sixstrains of recombinant Escherichia coli was successfully built, and selected the E. coli BL21(DE3)/pET28a-plcH as major research strain from recombinant strains. The recombinantstrain showed strong PLC activity and hemolytic activity on yolk borax plate and Columbiablood agar plate.The optimal fermentation conditions for E. coli BL21(DE3)/pET28a-plcH in LB mediumwere obtained with5%of inoculation amount,200r/min for4hours at temperature of37,induced by0.9mmol/L IPTG or0.05g/L lactose for14hours. The enzyme activity of PLCHwas reached to722.89±0.47U/mL or823.17±38.27U/mL under these conditions.The optimal fermentation conditions for E. coli BL21(DE3)/pET28a-plcH in LBmedium by added lactose and glycine were obtained with5%of inoculation amount,200r/min for4hours at temperature of37, induced by0.05g/L lactose for14hours, and thenadded15.0g/L glycine, induced by continued to48h, the enzyme activity of PLCH reachedto3676±55.19U/mL.This phenomenon showed that glycine could promote PLCH productionin LB medium.The optimal fermentation conditions for E. coli BL21(DE3)/pET28a-plcH in TB-THmedium were obtained with5%of inoculation amount,200r/min for6hours at temperatureof37, induced by0.6mmol/L IPTG or5g/L lactose for20or22hours. The enzyme activityof PLCH was up to852.91±24.84U/mL or1422.42±37.17U/mL under these conditions. Theresults showed that glycine could significant inhibit the production of PLCH in TB-THmedium.The recombinant E. coli BL21(DE3)/pET28a-plcH was subjected to fermentation in a7L fermenter using an optimized TB medium. The results showed that the enzyme activity ofPLCH was reached to11583.35±70.21U/mL when individually adding lactose.