High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

Abstract The acetylcholinesterase 2 ( AChE2 ) cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115. One transformant with high-level expression of the recombinant AChE (rAChE, 23.2 U mL −1 in supernatant) was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate. The maximum production of rAChE reached about 11.8 mg of the enzyme protein per liter of culture. The rAChE was first precipitated with ammonium sulfate (50% saturation) and then purified with procainamide affinity column chromatography. The enzyme was purified 12.1-fold with a yield of 22.8% and a high specific activity of 448.3 U mg −1 . It was sensitive to inhibition by methamidophos and pirimicarb, the calculated 50% inhibitory concentration (IC 50 ) values of the two pesticides were 0.357 and 0.888 mg L −1 , respectively, and the calculated 70% inhibitory concentration (IC 70 ) values were 0.521 and 0.839 mg L −1 , respectively. The results suggested that it has a potential application in the detection of pesticide residues.