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 (hBChE) 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 mL–1 with an activity of 14.7 U mL–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 hBChE 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 bimolecular 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 than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.

    Increases in the number of cases of identified genetically modified (GM) rice contamination can be traced back to the first Rapid Alert System for Food and Feed (RASFF) in 2006. In response to the lack of reliable detection methods, Decision 2011/884/EU proposed that new screening methods replace Decision 2008/289/EC, to identify all possible GM rice products originating in China. However, the synergy brands (SYBR) Green real-time PCR assay proposed by Decision 2011/884/EU has been shown to lack conformity with other TaqMan methods currently in use. To evaluate the specificity and repeatability of the methods recommended in Decision 2011/884/EU and Decision 2008/289/EC, we collected 74 rice products originating from six countries or districts. The 74 rice samples were tested using the Decision 2011/884/EU and Decision 2008/289/EC methods. The parallel use of different instruments and reagents were used for testing in parallel, and the results were analyzed statistically. To avoid the limitations of specific laboratories, eight GM organism detection laboratories in China participated in a collaborative trial. In our tests, 24.3% (18/74) of the samples tested were positive with the SYBR Green real-time PCR assay using the Decision 2011/884/EU method, but were negative with the TaqMan real-time PCR assay using the Decision 2011/884/EU and Decision 2008/289/EC methods. Sequencing the PCR-amplified CryIA(b/c) genes in three samples (6, 30 and 43) showed that the products consisted of primer dimers rather than the targeted sequence. The combined experimental results showed that testing for the nopaline synthase gene (NOS) of Agrobacterium tumefasciens terminator and CryIA(b/c) produced false-positive results when the Decision 2011/884/EU method was used. Because of the high rate of false-positive results, the Decision 2011/884/EU SYBR Green method to detect GM rice requires improvement.