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| Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues |
| TIAN Jing-jing, CHEN Xiang-ning, XIE Yuan-hong, LU Yong, XU Wen-tao, XU Li, DU Bin |
1、College of Food Science and Engineering, Beijing University of Agriculture, Beijing 102206, P.R.China
2、Beijing Food Safety Monitoring Center, Beijing 100041, P.R.China
3、College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R.China |
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摘要 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.
Abstract 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.
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Received: 18 December 2014
Accepted:
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| Fund: This work was financially supported by the Rural Work Committee of Beijing City for the First Batch of Agricultural Science and Technology Project of Beijing University of Agriculture, China (2013010102), the Beijing Innovation Team Building Project of Leafy Vegetables of Modern Agricultural Industry System, China (2063213003), and the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions, China (CIT&TCD 20150315). |
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Corresponding Authors:
CHEN Xiang-ning, Tel/Fax: +86-10-80799174, E-mail: chenxiangning@bua.edu.cn
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| About author: TIAN Jing-jing, Mobile: +86-18811582208, E-mail: 416718211@qq.com; |
Cite this article:
TIAN Jing-jing, CHEN Xiang-ning, XIE Yuan-hong, LU Yong, XU Wen-tao, XU Li, DU Bin.
2016.
Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues. Journal of Integrative Agriculture, 15(3): 684-693.
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