Purification and some characteristics of recombinant insecticide-resistant mosquito carboxylesterase B1 expressed in Escherichia coli

A recombinant insecticide-resistant mosquito carboxylesterase B1 was purified to homogeneity from an Escherichia coli expression system. After non-denaturing electrophoresis, active carboxylesterase B1 bands were identified using fast blue RR. Lineweaver–Burk plots of the crude and purified CaE B1 indicate that this enzyme obeys Michaelis–Menten kinetics with Km value for malathion of 39.3 and 67.4 mM. The Vm of purified enzyme is approximately 17-folds of the value determined in crude homogenate. Carboxylesterase B1 detoxification of parathion had a major limitation which is the 1:1 stoichiometry. To improve the effectiveness of enzymatic detoxification, we developed an approach in which the catalytic activity of organophosphorus compound-inhibited carboxylesterase B1 was restored by having sufficient amounts diacetylmonoxime. It was demonstrated that repeated addition of 25 times the molar concentration of parathion to carboxylesterase B1 in the presence of 4 mM diacetylmonoxime every 2 h did not result in significant inhibition of the enzyme. Consequently the stoichiometry of enzyme detoxification is higher than 64: 1 for parathion.