The cholinesterase-based spectrophotometric assay, also called enzyme inhibition method, is a good choice for rapid detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs). Obviously, the cholinesterase is the core reagent in enzyme inhibition method. In our previous work, a recombinant acetylcholinesterase 2 from Bombyx mori (rBmAChE2) was expressed in yeast successfully and exhibited great sensitivity. However, the yield of rBmAChE2 is not desirable. In this study, a codon optimization strategy was employed to enhance the yield of rBmAChE2 in Pichia pastoris GS115. Results showed that by replacing 6 key rare codons and increasing the percentage of bases G and C up to 46.85%, codon adaptation index (CAI) of Bombyx mori acetylcholinesterase 2 (bmace2) gene was improved from 0.70 to 0.81. After being transformed into Pichia pastoris GS115 via electroporation, the expression transformant can produce 139.7 U/mL secretory codon-optimized rBmAChE2 (opt-rBmAChE2) in the culture supernatant, 3.62 times higher than that of strain bearing the wild-type bmace2 gene. Meanwhile, opt-rBmAChE2 displayed on the yeast surface was up to 2280.02 U/g, 2.8 times higher than wild-type displayed rBmAChE2. In addition, either secretory or surface-displayed opt-rBmAChE2 maintained the similar sensitivities to the wild-type rBmAChE2 for tested inhibitors. Furthermore, the detection limits of the opt-rBmAChE2-based enzyme inhibition method for 10 kinds of OPs or CPs (0.01-2.69 mg/kg) were lower than most of the indexes present in current standard method (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019) in China. The results might contribute to the utilization of rBmAChE2 for pesticide residue screening detection in practice.
Keywords: Acetylcholinesterase; Codon optimization; Pesticides; Pichia pastoris.
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