Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Abstract

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.

Molecular characterization and inhibition analysis of the acetylcholinesterase gene from the silkworm maggot, Exorista sorbillans

“…The affinity column was prepared using our described method (17) with ECH Sepharose 4B as a matrix and procainamide as a ligand. Protein concentrations were determined by the Bradford method using a bovine serum albumin (BSA) as a standard and measuring OD at 595 nm, using a microplate reader.…”

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS

“…Acetylcholinestrase (AChE, EC 3.1.1.7) is an important serine hydrolase. It catalyzes hydrolysis of the neurotransmitter acetylcholine (Ach) during nerve conduction to acetic acid and choline, maintaining normal nerve conduction (Lang et al., ). AChE is also a target of organophosphate (OP) and carbamate (CB) insecticides, which irreversibly bind to and inhibit AChE activity, resulting nerve disorders and death (Nath and Kumar, ).…”

Functional study on the mutations in the silkworm (Bombyx mori) acetylcholinesterase type 1 gene (ace1) and its recombinant proteins

“…AChE is the target enzyme for organophosphorus and carbamate pesticides [1]. These pesticides can bind to AChE and reduce its activity, leading to massive accumulation of postsynaptic membrane acetylcholine, continuous stimulation, biological convulsion and the eventual insect death.…”