Abstract
Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophila melanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL·min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.
摘要
目的
通过比较果蝇乙酰胆碱酯酶基因剪接变种(DmAChE-like)与果蝇乙酰胆碱酯酶(DmAChE)之间的酶活差异以及对有机磷农药的敏感性差异,为酶抑制法检测农药残留提供新的酶源。
创新点
DmAChE-like 是果蝇乙酰胆碱酯酶基因发生剪接突变产生的,在此之前并未被报道过. 本文通过检测证明了DmAChE-like具有乙酰胆碱酯酶活性,并且对大多数有机磷农药敏感. 这个发现为酶抑制法检测农药残留提供了新的酶源.。这个发现为酶抑制法检测农药残留提供了新的酶源。
方法
在本研究中,我们通过构建毕赤酵母重组表达菌株GS115-pPIC9K-DmAChE-like 并进行诱导表达,利用Ellman法检测其酶活,并进行酶学性质研究。我们选取了5种常见农药进行农药敏感性试验,比较其与DmAChE之间的差异性。
结论
在本研究中,我们构建了基因工程的GS115-pPIC9K-DmAChE-like 菌株,并在实验室水平诱导了基因表达。我们测试了各个发酵上清液的酶活性,证明了DmAChE-like具有乙酰胆碱酯酶活性。通过Tricine-SDS-PAGE,我们发现特异的蛋白条带为4.3 kDa,与预期结果一致。农药敏感性试验发现DmAChE-like对大多数选择的有机磷农药敏感,表明它可以用作有机磷农药检测酶抑制方法的新酶源。
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References
Albero B, Sánchez-Brunete C, Tadeo JL, 2003. Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography. J Agric Food Chem, 51(24):6915–6921. https://doi.org/10.1021/jf030414m
Aldridge WN, Davison AN, 1953. The mechanism of inhibition of cholinesterases by organophosphorus compounds. Biochem J, 55(5):763–766. https://doi.org/10.1042/bj0550763
Amine A, Mohammadi H, Bourais I, et al., 2006. Enzyme inhibition-based biosensors for food safety and environmental monitoring. Biosens Bioelectron, 21(8):1405–1423. https://doi.org/10.1016/j.bios.2005.07.012
Apilux A, Isarankura-Na-Ayudhya C, Tantimongcolwat T, et al., 2015. Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection. EXCLI J, 14:307–319. https://doi.org/10.17179/excli2014-684
Chen DQ, Chen CHZ, Du D, 2010. Detection of organophosphate pesticide using polyaniline and carbon nanotubes composite based on acetylcholinesterase inhibition. J Nanosci Nanotechnol, 10(9):5662–5666. https://doi.org/10.1166/jnn.2010.2477
Duford DA, Xi YQ, Salin ED, 2013. Enzyme inhibition-based determination of pesticide residues in vegetable and soil in centrifugal microfluidic devices. Anal Chem, 85(16):7834–7841. https://doi.org/10.1021/ac401416w
Ellman GL, Courtney KD, Andres V, et al., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol, 7(2):88–95. https://doi.org/10.1016/0006-2952(61)90145-9
Fournier D, Mutero A, 1994. Modification of acetylcholinesterase as a mechanism of resistance to insecticides. Comp Biochem Physiol C Pharmacol Toxicol Pharmacol Endocrinol, 108(1):19–31. https://doi.org/10.1016/1367-8280(94)90084-1
Goujon M, McWilliam H, Li WZ, et al., 2010. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res, 38(suppl_2):W695–W699. https://doi.org/10.1093/nar/gkq313
Hossain MS, Sikder MT, 2015. Potential human health impacts and medical treatment of acute poisoning with organophosphorus pesticides (OPs): a review. Int J Environ Prot Policy, 3(2):6–13. https://doi.org/10.11648/j.ijepp.s.2015030201.12
Jokanoviý M, 2009. Medical treatment of acute poisoning with organophosphorus and carbamate pesticides. Toxicol Lett, 190(2):107–115. https://doi.org/10.1016/j.toxlet.2009.07.025
Koureas M, Tsakalof A, Tsatsakis A, et al., 2012. Systematic review of biomonitoring studies to determine the association between exposure to organophosphorus and pyrethroid insecticides and human health outcomes. Toxicol Lett, 210(2):155–168. https://doi.org/10.1016/j.toxlet.2011.10.007
Lang QL, Han L, Hou CT, et al., 2016. A sensitive acetylcholinesterase biosensor based on gold nanorods modified electrode for detection of organophosphate pesticide. Talanta, 156–157:34–41. https://doi.org/10.1016/j.talanta.2016.05.002
Li JQ, Ba Q, Yin J, et al., 2013. Surface display of recombinant Drosophila melanogaster acetylcholinesterase for detection of organic phosphorus and carbamate pesticides. PLoS ONE, 8(9):e72986. https://doi.org/10.1371/annotation/5c68b3d3-240f-4e87-b394-ca5301479cef
Liang B, Wang G, Yan L, 2019. Functional cell surface displaying of acetylcholinesterase for spectrophotometric sensing organophosphate pesticide. Sens Actuators B Chem, 279:483–489. https://doi.org/10.1016/j.snb.2018.09.119
Mai CQ, Chen S, Chen Y, 2017. Enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. Plant Dis Pests, 8(4):30–32. https://doi.org/10.19579/j.cnki.plant-d.p.2017.04.008
Nielsen H, Engelbrecht J, Brunak S, et al., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng, 10(1):1–6. https://doi.org/10.1093/protein/10.1.1
Pardo MF, Natalucci CL, 2002. Electrophoretic analysis (tricine-SDS-PAGE) of bovine caseins. Acta Farm Bonaerense, 21(1):57–60.
Petersen TN, Brunak S, von Heijne G, et al., 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods, 8(10):785–786. https://doi.org/10.1038/nmeth.1701
Ponomarev AS, Shtykov SN, 2000. Determination of pesticides and other physiologically active compounds by capillary gas chromatography with an atomic-emission detector without using standard reference materials. J Anal Chem, 55(1):47–51. https://doi.org/10.1007/BF02757632
Pope CN, 1999. Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health B Crit Rev, 2(2):161–181. https://doi.org/10.1080/109374099281205
Sakata M, 1993. Organophosphorus pesticides. In: Suzuki O, Watanabe K (Eds.), Drugs and Poisons in Humans. Springer, Berlin, Heidelberg. https://doi.org/10.1007/3-540-27579-7_60
Schägger H, 2006. Tricine-SDS-PAGE. Nat Protoc, 1(1):16–22. https://doi.org/10.1038/nprot.2006.4
Schulze H, Muench SB, Villatte F, et al., 2005. Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B. Anal Chem, 77(18): 5823–5830. https://doi.org/10.1021/ac050383p
Tang XJ, Liang B, Yi TY, et al., 2014a. Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates. Enzyme Microb Technol, 55:107–112. https://doi.org/10.1016/j.enzmictec.2013.10.006
Tang XJ, Zhang TT, Liang B, et al., 2014b. Sensitive electrochemical microbial biosensor for p-nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons. Biosens Bioelectron, 60:137–142. https://doi.org/10.1016/j.bios.2014.04.001
Tougu V, 2001. Acetylcholinesterase: mechanism of catalysis and inhibition. Curr Med Chem Cent Nerv Syst Agents, 1(2): 155–170. https://doi.org/10.2174/1568015013358536
van Dyk JS, Pletschke B, 2011. Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment. Chemosphere, 82(3):291–307. https://doi.org/10.1016/j.chemosphere.2010.10.033
Villatte F, Marcel V, Estrada-Mondaca S, et al., 1998. Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides. Biosens Bioelectron, 13(2):157–164. https://doi.org/10.1016/S0956-5663(97)00108-5
Viñas P, Campillo N, López-García I, et al., 2003. Capillary gas chromatography with atomic emission detection for pesticide analysis in soil samples. J Agric Food Chem, 51(13):3704–3708. https://doi.org/10.1021/jf021106b
Worek F, Eyer P, Thiermann H, 2012. Determination of acetylcholinesterase activity by the ellman assay: a versatile tool for in vitro research on medical countermeasures against organophosphate poisoning. Drug Test Anal, 4(3–4): 282–291. https://doi.org/10.1002/dta.337
Yang X, Dai J, Yang L, et al., 2018. Oxidation pretreatment by calcium hypochlorite to improve the sensitivity of enzyme inhibition-based detection of organophosphorus pesticides. J Sci Food Agric, 98(7):2624–2631. https://doi.org/10.1002/jsfa.8755
Yang XM, Gu YP, Wu SJ, et al., 2019. Research on a rapid detection method of pesticide residues in milk by enzyme inhibition. E3S Web Conf, 79:03013. https://doi.org/10.1051/e3sconf/20197903013
Zhou WJ, Yang JK, Mao L, et al., 2015. Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris. Enzyme Microb Technol, 71:66–72. https://doi.org/10.1016/j.enzmictec.2014.10.007
Acknowledgments
This work was supported by the National Development and Reform Commission of China (No. 20111158). We thank Wentao JING Engineer (Biological Experiment Teaching Center, College of Life Sciences Zhejiang University Hangzhou China) for providing the RNA samples from the head of Drosophila melanogaster.
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Liujia SHI performed the experimental research and data analysis, wrote and edited the manuscript. Fangfang YANG discovered the DmAChE-like gene and verified its function. Yanyan XU provided assistance for the experiment. Shoufeng WANG contributed to the study design, data analysis, and writing and editing of the manuscript. All authors have read and approved the final manuscript and, and therefore have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Liujia SHI, Fangfang YANG, Yanyan XU, and Shoufeng WANG declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Shi, L., Yang, F., Xu, Y. et al. Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides. J. Zhejiang Univ. Sci. B 22, 204–213 (2021). https://doi.org/10.1631/jzus.B2000525
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DOI: https://doi.org/10.1631/jzus.B2000525