Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues

Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase（BChE, EC 3.1.1.8）, which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene（h BCh E） as published. The ratio of guanine and cytosine in four kinds of bases（（G+C） ratio） was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L–1 with an activity of 14.7 U m L–1, which was purified 3.2×103-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range（>60% activity at pH 4.0 to 8.0）. Moreover, it had a good adaptability to high temperature（>60% activity at both 50 and 60°C up to 60 min） and good stability at 70°C. The enzyme can be activated by Li+, Co+, Zn2+ and ethylene diamine tetraacetic acid（EDTA）, but inhibited by Mg2+, Mn2+, Fe2+, Ag+ and Ca2+. Na+ had little effect on its activity. The values of h BChE of the Michaelis constant（Km） and maximum reaction velocity（Vm） were 89.4 mmol L–1 and 1 721 mmol min–1 mg–1, respectively. The bim olecular rate constants（Ki） of the hBChE to four pesticides were similar with that of electric eel AChE（EeAChE） and higher than that of horse BChE（HoBChE）. All values of the half maximal inhibitory concentration of a substance(IC50) for hBChE were lower th