Cloning&Expression And Application Of Carboxylesterase Gene

Gene cloning, expression, characterization and degradation of carboxylesterase from mouse liver were explored in this paper. The ceslf gene coding carboxylesterase was amplified by RT-PCR with the total RNA of Mouse liver as the template. The ceslf were inserted in pUCm-T to construct the recombinant cloning vector pUCm-T-ceslf. After the DNA sequence was determined, the ceslf was subcloned into expression vector pET-32a to construct the recombinant expression vector pET-32a-ceslf, and transformed into the host strain E.coli BL21(DE3). Upon IPTG induction, the SDS-PAGE analysis showed a band with apparent molecular weight of77kDa, but was expressed in the form of inclusion body.The best expression condition with culture pH6.5, temperature20℃,inoculum-size3:200,IPTG concentration0.6mM,and culture time8h were decided. Recombinant purified by Ni-NTA affinity chromatography, its specified activity was about55.37U/mg.The Km of the recombinant for a-Naphthylacetate,β-Naphthylacetate as substrate were0.06125mMå'Œ0.1334mM respectively, the optimal substrate was a-Naphthylacetate. The optimal temperature was27℃and the optimal pH was pH6.0-pH7.0. HPLC detection showed carbaryl, carbofuran, metaphos and bifenthrin were degraded by the recombinant, laying a foundation for the degradation of residual pesticides in environment.