Bacterial Expression and Kinetic Analysis of Carboxylesterase 001D from Helicoverpa armigera

Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup ofHelicoverpa armigeraesterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene001DofH. armigerafrom China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D inEscherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with aKcat between 0.35 and 2.29 s−1, and aKm between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towardsβ-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min−1·(μM−1·protein)). The enzyme was stable up to 40 °C and at pH 6.0–11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides inH. armigera.Keywords:Helicoverpaarmigera;carboxylesterase;pyrethroid;heterologous expression