[Cloning and fusion expression of detoxifying gene in Escherichia coli]

In insects, esterases play an important role in the degradation of organophosphate (OP) and Carbamate (CB) insecticides. The ability of esterase B1 to degrading OP and CB insecticides opens broad prospect of using it in programs to the insecticide pollution. OPs can be detoxified by hydrolysis of their phosphoester bonds, pyrethroids and some OPs like malathion, by hydrolysis of their carboxylester bonds, and diverse carbamate insecticides, herbicides and fungicides by hydrolysis of amide or other similar bonds. Most of the candidate bioremediation enzymes identified to date have been hydrolases. In recent years, we have focused our work on the transfer of a detoxifying esterase B1 gene from mosquito into E. coli and explored the possibility of using the detoxified enzymes in environmental protection. 5' initial of esterase B1 cDNA was cloned by RT-PCR and sequenced consequently. After the combination of 5' initial and 3' terminal fragment of esterase B1 cDNA, the recombinant vector pET-ESTB1 was constructed and transformed into E. coli BL21. A 60 kD protein was induced by IPTG and its expression was temperature-dependent. After 12 h induction, the target protein occupied 27% of the total protein. A pure recombinant protein was obtained by purification, and was detected with 10% SDS-PAGE. The results showed that 22.1% of malathion was degraded by crude detoxification enzyme in 15 mins, demonstrated a high degradation property. This research provides a novel approach, which takes the advantages of eucaryotes for bioremediation of pesticide pollutions.