|
|
|
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 |
|
|
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.
|
Received: 18 December 2014
Accepted:
|
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). |
Corresponding Authors:
CHEN Xiang-ning, Tel/Fax: +86-10-80799174, E-mail: chenxiangning@bua.edu.cn
E-mail: chenxiangning@bua.edu.cn
|
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.
|
Alber R, Sporns O, Weikert T, Willbold E, Layer P. 1994.Cholinesterases and peanut agglutinin binding related tocell proliferation and axonal growth in embryonic chick limbs.Anatomy and Embryology, 190, 429-438Altshull S, Madden T, Schäffer A, Zhang J, Zhang Z, MillerW, Lipman D. 1997. Gapped blast and psiblast: A newgeneration of protein database search programs. NucleicAcids Research, 25, 3389-3402Boublik Y, Saint-Aguet P, Lougarre A, Arnaud M, Villatte F,Estrada-Mondaca S, Fournier D. 2002. Acetylcholinesteraseengineering for detection of insecticide residues. ProteinEngineering, 15, 43-50Bradford M M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Analytical Biochemistry,72, 248-254Chilukuri N, Sun W, Naik R, Parikh K, Tang L, Doctor B, SaxenaA. 2008. Effect of polyethylene glycol modification on thecirculatory stability and immunogenicity of recombinanthuman butyrylcholinesterase. Chemico-BiologicalInteractions, 175, 255-260Ellman G, Courtney K, Andres V, Featherstone R. 1961. A newand rapid colorimetric determination of acetylcholinesteraseactivity. Biochemical Pharmacology, 7, 88-95Graham S, Francis Y, Choy M. 2002. Synonymous codon usagebias and the expression of human glucocerebrosidase in themethylotrophic yeast, Pichia pastoris. Protein Expressionand Purification, 26, 96-105Harel M, Sussman J, Krejci E, Bon S, Chanal P, MassoulieJ, Silman I. 1992. Conversion of acetylcholinesteraseto butyrylcholinesterase: Modeling and mutagenesis.Proceedings of the National Academy of Sciences of theUnited States of America, 89, 10827-10831Ilyushin D, Haertley O, Bobik T, Shamborant O, Surina E, KnorreV, Masson P, Smirnov I, Gabibov A, Ponomarenko N. 2013.Recombinant human butyrylcholinesterase as a new-agebioscavenger drug: Development of the expression system.Acta Naturae, 5, 73.Jain S, Hreczuk-Hirst D, McCormack B, Mital M, EpenetosA, Laing P, Gregoriadis G. 2003. Polysialylated insulin:Synthesis, characterization and biological activity in vivo.Biochimica et Biophysica Acta (BBA)-General Subjects,1622, 42-49Jia H, Fan G, Yan Q, Liu Y, Yan Y, Jiang Z. 2012. High-levelexpression of a hyperthermostable Thermotoga maritimaxylanase in Pichia pastoris by codon optimization. Journalof Molecular Catalysis (B: Enzymatic), 78, 72-77Li B, Stribley J, Ticu A, Xie W, Schopfer L, Hammond P,Brimijoin S, Hinrichs S, Lockridge O. 2000. Abundanttissue butyrylcholinesterase and its possible function inthe acetylcholinesterase knockout mouse. Journal ofNeurochemistry, 75, 1320-1331Lockridge O. 1990. Genetic variants of human serumcholinesterase influence metabolism of the muscle relaxantsuccinylcholine. Pharmacology & Therapeutics, 47, 35-60Macauley-Patrick S, Fazenda M, McNeil B, Harvey L. 2005.Heterologous protein production using the Pichia pastorisexpression system. Yeast, 22, 249-270Massoulié J, Pezzementi L, Bon S, Krejci E, Vallette F. 1993.Molecular and cellular biology of cholinesterases. Progressin Neurobiology, 41, 31-91Mattes C, Lynch T, Singh A, Bradley R, Kellaris P, Brady R,Dretchen K. 1997. Therapeutic use of butyrylcholinesterasefor cocaine intoxication. Toxicology and AppliedPharmacology, 145, 372-380Mchunu N, Singh S, Permaul K. 2009. Expression of analkalo-tolerant fungal xylanase enhanced by directedevolution in Pichia pastoris and Escherichia coli. Journalof Biotechnology, 141, 26-30Mendelsohn R, Nordhaus W, Shaw D. 1994. The impact ofglobal warming on agriculture: A Ricardian analysis. TheAmerican Economic Review, 84, 753-771Mesulam M M, Guillozet A, Shaw P, Levey A, Duysen E, LockridgeO. 2002. Acetylcholinesterase knockouts establish centralcholinergic pathways and can use butyrylcholinesteraseto hydrolyze acetylcholine. Neuroscience, 110, 627-639Ordentlich A, Barak D, Kronman C, Flashner Y, Leitner M,Segall Y, Ariel N, Cohen S, Velan B, Shafferman A. 1993.Dissection of the human acetylcholinesterase activecenter determinants of substrate specificity. Identificationof residues constituting the anionic site, the hydrophobicsite, and the acyl pocket. Journal of Biological Chemistry,268, 17083-17095Pritchard J, Law K, Vakurov A, Millner P, Higson S. 2004.Sonochemically fabricated enzyme microelectrode arraysfor the environmental monitoring of pesticides. Biosensorsand Bioelectronics, 20, 765-772Radic Z, Pickering N, Vellom D, Camp S, Taylor P. 1993. Threedistinct domains in the cholinesterase molecule conferselectivity for acetyl- and butyrylcholinesterase inhibitors.Biochemistry, 32, 12074-12084Sasaki K, Suzuki T, Saito Y. 1987. Simplified cleanup andgas chromatography determination of organophosphoruspesticides in crops. Journal of the Association of OfficialAgriculture Chemists, 70, 460-465 Sinclair G, Choy F. 2002. Synonymous codon usage biasand the expression of human glucocerebrosidase in themethylotrophic yeast, Pichia pastoris. Protein Expressionand Purification, 26, 96-105Small D, Michaelson S, Sberna G. 1996. Non-classicalactions of cholinesterases: Role in cellular differentiation,tumorigenesis and Alzheimer’s disease. NeurochemistryInternational, 28, 453-483Taylor P, Radic Z. 1994. The cholinesterases: From genes toproteins. Annual Review of Pharmacology and Toxicology,34, 281-320Vakurov A, Simpson C, Daly C, Gibson T, Millner P. 2004.Acetylcholinesterase-based biosensor electrodes fororganophosphate pesticide detection: I. Modification ofcarbon surface for immobilization of acetylcholinesterase.Biosensors and Bioelectronics, 20, 1118-1125Wang H, Wang Q, Zhang F, Huang Y, Ji Y, Hou Y. 2008. Proteinexpression and purification of human Zbtb7A in Pichiapastoris via gene codon optimization and synthesis. ProteinExpression and Purification, 60, 97-102Wei W, Zhang H, Qin J, Sun M. 1998. Characteristics ofrecombinant human butyrylcholinesterase expressedin Bombyx mori expression system. Chinese Journal ofBiochemistry and Molecular Biology, 15, 797-801 (inChinese)Zhu H, Li X, Deng Y, Wang S, Fu W, Tang L, Gao Z,Tang Y. 2011. Purification and characterization of thebutyrylcholinesterase from duck liver. Science andTechnology of Food Industry, 1, 95–99. (in Chinese) |
No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|