Full Text:   <925>

Summary:  <941>

CLC number: 

On-line Access: 2021-03-12

Received: 2020-09-04

Revision Accepted: 2020-11-16

Crosschecked: 2021-01-29

Cited: 0

Clicked: 1619

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Liujia SHI

https://orcid.org/0000-0001-5124-7845

Shoufeng WANG

https://orcid.org/0000-0003-0896-5440

-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2021 Vol.22 No.3 P.204-213

http://doi.org/10.1631/jzus.B2000525


Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides


Author(s):  Liujia SHI, Fangfang YANG, Yanyan XU, Shoufeng WANG

Affiliation(s):  Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou 310058, China; more

Corresponding email(s):   sfwang@zju.edu.cn

Key Words:  Acetylcholinesterase (AChE), Pichia pastoris, Enzyme activity determination, Pesticide sensitivity


Liujia SHI, Fangfang YANG, Yanyan XU, Shoufeng WANG. Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides[J]. Journal of Zhejiang University Science B, 2021, 22(3): 204-213.

@article{title="Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides",
author="Liujia SHI, Fangfang YANG, Yanyan XU, Shoufeng WANG",
journal="Journal of Zhejiang University Science B",
volume="22",
number="3",
pages="204-213",
year="2021",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2000525"
}

%0 Journal Article
%T Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides
%A Liujia SHI
%A Fangfang YANG
%A Yanyan XU
%A Shoufeng WANG
%J Journal of Zhejiang University SCIENCE B
%V 22
%N 3
%P 204-213
%@ 1673-1581
%D 2021
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2000525

TY - JOUR
T1 - Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides
A1 - Liujia SHI
A1 - Fangfang YANG
A1 - Yanyan XU
A1 - Shoufeng WANG
J0 - Journal of Zhejiang University Science B
VL - 22
IS - 3
SP - 204
EP - 213
%@ 1673-1581
Y1 - 2021
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2000525


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.

果蝇乙酰胆碱酯酶基因剪接变种在毕赤酵母中的表达及其对有机磷农药敏感性的评价

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。
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。

关键词:乙酰胆碱酯酶;巴斯德毕赤酵母;酶活性测定;农药敏感性

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1]AlberoB, Sánchez-BruneteC, TadeoJL, 2003. Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography. J Agric Food Chem, 51(24):6915-6921.

[2]AldridgeWN, DavisonAN, 1953. The mechanism of inhibition of cholinesterases by organophosphorus compounds. Biochem J, 55(5):763-766.

[3]AmineA, MohammadiH, BouraisI, et al., 2006. Enzyme inhibition-based biosensors for food safety and environmental monitoring. Biosens Bioelectron, 21(8):1405-1423.

[4]ApiluxA, Isarankura-Na-AyudhyaC, TantimongcolwatT, et al., 2015. Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection. EXCLI J, 14:307-319.

[5]ChenDQ, ChenCHZ, DuD, 2010. Detection of organophosphate pesticide using polyaniline and carbon nanotubes composite based on acetylcholinesterase inhibition. J Nanosci Nanotechnol, 10(9):5662-5666.

[6]DufordDA, XiYQ, SalinED, 2013. Enzyme inhibition-based determination of pesticide residues in vegetable and soil in centrifugal microfluidic devices. Anal Chem, 85(16):7834-7841.

[7]EllmanGL, CourtneyKD, AndresV, et al., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol, 7(2):88-95.

[8]FournierD, MuteroA, 1994. Modification of acetylcholinesterase as a mechanism of resistance to insecticides. Comp Biochem Physiol C Pharmacol Toxicol Pharmacol Endocrinol, 108(1):19-31.

[9]GoujonM, McWilliamH, LiWZ, et al., 2010. A new bioinformatics analysis tools framework at EMBL‒EBI. Nucleic Acids Res, 38(suppl_2):W695-W699.

[10]HossainMS, SikderMT, 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.

[11]JokanovićM, 2009. Medical treatment of acute poisoning with organophosphorus and carbamate pesticides. Toxicol Lett, 190(2):107-115.

[12]KoureasM, TsakalofA, TsatsakisA, 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.

[13]LangQL, HanL, HouCT, et al., 2016. A sensitive acetylcholinesterase biosensor based on gold nanorods modified electrode for detection of organophosphate pesticide. Talanta, 156-157:34-41.

[14]LiJQ, BaQ, YinJ, et al., 2013. Surface display of recombinant Drosophila melanogaster acetylcholinesterase for detection of organic phosphorus and carbamate pesticides. PLoS ONE, 8(9):e72986.

[15]LiangB, WangG, YanL, 2019. Functional cell surface displaying of acetylcholinesterase for spectrophotometric sensing organophosphate pesticide. Sens Actuators B Chem, 279:483-489.

[16]MaiCQ, ChenS, ChenY, 2017. Enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. Plant Dis Pests, 8(4):30-32.

[17]NielsenH, EngelbrechtJ, BrunakS, et al., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng, 10(1):1-6.

[18]PardoMF, NatalucciCL, 2002. Electrophoretic analysis (tricine-SDS-PAGE) of bovine caseins. Acta Farm Bonaerense, 21(1):57-60.

[19]PetersenTN, BrunakS, von HeijneG, et al., 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods, 8(10):785-786.

[20]PonomarevAS, ShtykovSN, 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.

[21]PopeCN, 1999. Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health B Crit Rev, 2(2):161-181.

[22]Sakata M, 1993. Organophosphorus pesticides. In: Suzuki O, Watanabe K (Eds.), Drugs and Poisons in Humans. Springer, Berlin, Heidelberg.

[23]SchäggerH, 2006. Tricine-SDS-PAGE. Nat Protoc, 1(1):16-22.

[24]SchulzeH, MuenchSB, VillatteF, et al., 2005. Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B. Anal Chem, 77(18):5823-5830.

[25]TangXJ, LiangB, YiTY, et al., 2014a. Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates. Enzyme Microb Technol, 55:107-112.

[26]TangXJ, ZhangTT, LiangB, 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.

[27]TouguV, 2001. Acetylcholinesterase: mechanism of catalysis and inhibition. Curr Med Chem Cent Nerv Syst Agents, 1(2):155-170.

[28]van DykJS, PletschkeB, 2011. Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment. Chemosphere, 82(3):291-307.

[29]VillatteF, MarcelV, Estrada-MondacaS, et al., 1998. Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides. Biosens Bioelectron, 13(2):157-164.

[30]ViñasP, CampilloN, López-GarcíaI, et al., 2003. Capillary gas chromatography with atomic emission detection for pesticide analysis in soil samples. J Agric Food Chem, 51(13):3704-3708.

[31]WorekF, EyerP, ThiermannH, 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.

[32]YangX, DaiJ, YangL, 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.

[33]YangXM, GuYP, WuSJ, et al., 2019. Research on a rapid detection method of pesticide residues in milk by enzyme inhibition. E3S Web Conf, 79:03013.

[34]ZhouWJ, YangJK, MaoL, 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.

Open peer comments: Debate/Discuss/Question/Opinion

<1>

Please provide your name, email address and a comment





Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2022 Journal of Zhejiang University-SCIENCE