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Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues
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作者 TIAN Jing-jing CHEN Xiang-ning +4 位作者 XIE Yuan-hong LU Yong XU Wen-tao XU Li DU Bin 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2016年第3期684-693,共10页
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 an... 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 3.2×10^3-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^+, Zn^2+ and ethylene diamine tetraacetic acid(EDTA), but inhibited by Mg^2+, Mn^2+, Fe^2+, Ag^+ and Ca^2+. 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(K_i) of the hBChE to four pesticides were similar with that of electric eel AChE(EeAChE) and higher than that of horse BChE(HoBChE). All vlues 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. 展开更多
关键词 butyrylcholinesterase organophosphate insecticide residues Pichia pastoris expression codon-optimized
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自动固相微萃取(SPM E)GC-MS、GC-MS-MS法检测环境水中有机磷杀虫剂的研究 被引量:6
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作者 魏立青 郭杰 +1 位作者 蒋华宇 种法运 《分析测试学报》 CAS CSCD 北大核心 2004年第z1期226-230,共5页
  固相微萃取(solid-phase microextraction,SPME)是20世纪90年代发展起来的一种样品前处理技术,与传统的液-液提取、液-固提取相比,SPME更适用于提取、浓缩液态或气态的挥发性和半挥发性物质,SPME技术可将采样、萃取、浓缩和样本引...   固相微萃取(solid-phase microextraction,SPME)是20世纪90年代发展起来的一种样品前处理技术,与传统的液-液提取、液-固提取相比,SPME更适用于提取、浓缩液态或气态的挥发性和半挥发性物质,SPME技术可将采样、萃取、浓缩和样本引入集中于一个步骤完成,尤其随着自动SPME与GC-MS等联用技术的日益完善,使SPME技术优点得到更充分的发挥.…… 展开更多
关键词 organophosphate insecticides Auto Solid-phase microextraction(auto-SPME) Gas chromatographymass spectrometry(GC - MS) Gas chromatography - multiple mass spectrometry(GC - MS - MS)
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Effects of Dimethoate Exposure on Locomotor Activity and Anxiety-Like Behavior in Female Wistar Rat
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作者 Majda Samih Pacome Kouadio N’Go +2 位作者 Sabah Belaaouja Amina Ouazzani Touhami Ahmed Omar Touhami Ahami 《Journal of Behavioral and Brain Science》 2017年第10期484-496,共13页
Developmental exposure to organophosphate insecticide is well known to induce neurobehavioral impairments, at late period. The present study aims to investigate the effects of subchronic exposure to Dimethoate, on loc... Developmental exposure to organophosphate insecticide is well known to induce neurobehavioral impairments, at late period. The present study aims to investigate the effects of subchronic exposure to Dimethoate, on locomotors skills and anxiety like behavior among wistar rat. Two groups of female’s rats are used. The intoxicated group receives daily, during five weeks, by intragastric gavage, a dose of Dimethoate dissolved in corn oil (100 mg/kg body weight). The control group receives only the corn oil. Spontaneous locomotors activity is evaluated using the Open Field test (OF) and anxiety-like behavior is measured using Elevated Plus-Maze (EPM). Dimethoate induced significant impairment of spontaneous locomotors activities, which is reflected by high decrease of number of squares crossed (SC) in OF. Females exposed to Dimethoate develop further anxiety-like response, expressed by significant reductions of the time spent in open arm of Elevated Plus-Maze. 展开更多
关键词 Acetylcholinesterase Activity NEUROTOXICITY DIMETHOATE NEUROBEHAVIOR organophosphate insecticides
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Developmental effects of Malathion exposure on locomotor activity and anxiety-like behavior in Wistar rat
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作者 Pacome Kouadio N’Go Fatima-Zahra Azzaoui +3 位作者 Ahmed Omar Touhami Ahami Porlo Rigobert Soro Mohamed Najimi Fatiha Chigr 《Health》 2013年第3期603-611,共9页
Developmental exposure to organophosphate insecticide is well known to induce neurobeha-vioral impairments, at late period. The present study aims to investigate the effects of chronic exposure to Malathion, from in u... Developmental exposure to organophosphate insecticide is well known to induce neurobeha-vioral impairments, at late period. The present study aims to investigate the effects of chronic exposure to Malathion, from in utero to young adult stage, on locomotor skills and anxiety like- behavior among wistar rat. Four groups of female rats, bred with one non-pesticide exposed male, are used. On gestational day 6, three groups receive daily, by intragastric gavage, 3 different doses of Malathion dissolved in corn oil (100, 200 and 300 mg/kg body weight). The control group receives the corn oil only. On postnatal day 21, weaned offsprings are submitted to the similar treatment until adult age. Spontaneous locomotor activity is evaluated using the Open-Field test (OF) and anxiety-like behavior is measured using both Open-Field (OF) test and Elevate Plus-Maze (EPM). Malathion at 300 mg/kg is toxic to pregnant dams, and pups are stillborns. In males, Malathionlevelat 100 and 200 mg/kg induced significant impairment of spontaneous locomotor activities, which is reflected by high decrease of number of squares crossed in OF. In contrast, no discernible changes are observed within females Malathion-treated-group. However, females exposed to both malathion levels develop further anxiety-like response, expressed by significant reductions of exploratory activities in OF and time spent in open arm of EPM. Neurochemistry assay shows that cerebellum and neocortex acetylcholinesterase (AChE) activity inhibition are significantly increased with neurobehavioral deficits in males, relative to females. Overall, neurobehavioral outcomes of current study reveal that developmental exposure to Malathion induces sex-selective effects with greater changes in females. 展开更多
关键词 AChE Activity Developmental Neurotoxicity MALATHION NEUROBEHAVIOR organophosphate insecticides
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