Study On High Cell Density Culture Of Recombinant Escherichia Coli For Pyrethroid Hydrolase Production

Pyrethroid insecticides with high insecticidal activity and low dosage have beenwidely used in modern agriculture. However, with the promotion and application ofpyrethroid pesticides in modern agriculture, pesticide residue has more serious impacton the environment. At present, there are physical, chemical and biological methodsto treat the residual of pyrethroid pesticide. The biological treatment, especiallyenzyme-degradation approach, can offer an efficient green solution to biodegradepolluting chemicals, thereby playing pivotal roles in the field of bioremediation.Pyrethroid hydrolase is a generic term, which cleavages the ester bond of thepyrethroid insecticides. At present, many pyrethroid hydrolases have been expressed,but most of studies about them stay on a low level of flask fermentation in thelaboratoryor or a low-density of fermentation stage in the high cell densityfermentation. And the yield of these pyrethroid hydrolases is low, which greatly limitsthe application of pyrethroid hydrolases in the environmental remediation. In order toimprove the yield of recombinant enzyme, high cell density culture to producepyrethroid hydrolases is urgently needed.Our research has been successfully constructed a recombinant Escherichia coliBL21/pET28a-sys410to express pyrethroid hydrolase before. The recombinant pyrethroid hydrolase plays good thermalstability and pH stability, which canefficiently degrade pyrethroid pesticides under normal temperature conditions. In thisstudy, a series of single-factor experiments and orthogonal test analysis in shake flaskwere studied, which demonstrated that the optimum medium was composed asfollows: glycerol20g/L, yeast extract20g/L, ammonium sulfate5g/L, phosphate100mmol/L, magnesium sulfate5mmol/L, trace element solution1.5ml/L. Underthe optimized conditions, the final cell density(OD₆₀₀)of the recombinant strainreached to10.4and the enzyme activity reached the maximum to97.4U/ml, which is1.8times and2times of those under the initial fermentation conditions, respectively.The high-density fermentation of recombinant strain in7.5L fermenters wasstudied. One process was obtained as follows: dispersed oxygen supply, lowerconcentration of the initial medium, and carbon and nitrogen sources in the Fed-batchprocess. The expression level of recombinant enzyme (OD₆₀₀=145) was about3000U/ml. The high-density fermentation of recombinant strain in30L and100Lfermenter was studied. The final cell density (OD₆₀₀) reached88and83,respectively. The enzyme activity reached1750U/ml and1565U/ml, respectively.In this study, the downstream processing of the recombinant enzyme was studied.Weight of enzyme powder by vacuum freeze-drying reached40g/L, and the enzymeactivity reached60000U/g.These results above showed that we has successfully produced the pyrethroidhydrolase with lower cost by high-density fermentation, which provided an effective,economic and feasible way for the mass production of pyrethroids hydrolase.