title: Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically modified Drosophila melanogaster: application in amperometric biosensors for methamidophos pesticide detection.
abstract: Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.
authors.affiliation: Núcleo de Análises de Resíduos de Pesticidas-NARP, Departamento de Tecnologia Química, CCET, Universidade Federal do Maranhão, Avenida dos Portugueses, s/n, CEP 65080-040 São Luís, MA, Brazil.
authors.forename: Paulo Roberto Brasil
authors.initials: PR
authors.lastname: de Oliveira Marques
authors.forename: Gilvanda Silva
authors.initials: GS
authors.lastname: Nunes
authors.forename: Teresa Cristina Rodrigues
authors.initials: TC
authors.lastname: dos Santos
authors.forename: Silvana
authors.initials: S
authors.lastname: Andreescu
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Marty
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000145:classification / Q000235:genetics*; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002417:Cattle; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004593:Electrophorus; D004784:Environmental Monitoring / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009946:Organothiophosphorus Compounds / Q000032:analysis*; D010575:Pesticides / Q000032:analysis; D015202:Protein Engineering / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D004800:Enzymes, Immobilized; D009946:Organothiophosphorus Compounds; D010575:Pesticides; C014655:methamidophos; D000110:Acetylcholinesterase