Engineering sensitive glutathione transferase for the detection of xenobiotics

dc.contributor.author	Kapoli, P	en
dc.contributor.author	Axarli, IA	en
dc.contributor.author	Platis, D	en
dc.contributor.author	Fragoulaki, M	en
dc.contributor.author	Paine, M	en
dc.contributor.author	Hemingway, J	en
dc.contributor.author	Vontas, J	en
dc.contributor.author	Labrou, NE	en
dc.date.accessioned	2014-06-06T06:48:27Z
dc.date.available	2014-06-06T06:48:27Z
dc.date.issued	2008	en
dc.identifier.issn	09565663	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.bios.2008.06.037	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/4158
dc.subject	Glutathione transferase	en
dc.subject	Ligandin binding site	en
dc.subject	Malathion	en
dc.subject	Potentiometric assay	en
dc.subject	Xenobiotics	en
dc.subject.other	Aldehydes	en
dc.subject.other	Arsenic compounds	en
dc.subject.other	Binding energy	en
dc.subject.other	Binding sites	en
dc.subject.other	Biochemistry	en
dc.subject.other	Chlorine compounds	en
dc.subject.other	Concentration (process)	en
dc.subject.other	Electrolysis	en
dc.subject.other	Enzymes	en
dc.subject.other	Food additives	en
dc.subject.other	Herbicides	en
dc.subject.other	Hydrophobicity	en
dc.subject.other	Insecticides	en
dc.subject.other	Metallizing	en
dc.subject.other	Pesticides	en
dc.subject.other	Potentiometric sensors	en
dc.subject.other	System theory	en
dc.subject.other	Technology	en
dc.subject.other	Bio sensor	en
dc.subject.other	Buffer systems	en
dc.subject.other	Calibration curves	en
dc.subject.other	Concentration ranges	en
dc.subject.other	Cytosolic	en
dc.subject.other	Free enzymes	en
dc.subject.other	Glutaraldehyde	en
dc.subject.other	Glutathione	en
dc.subject.other	Glutathione transferase	en
dc.subject.other	Glutathione transferases	en
dc.subject.other	High-capacity	en
dc.subject.other	Hydrophobic compounds	en
dc.subject.other	Immobilized enzymes	en
dc.subject.other	In-silico	en
dc.subject.other	Ligand-binding proteins	en
dc.subject.other	Ligandin binding site	en
dc.subject.other	Malathion	en
dc.subject.other	Molecular modelling	en
dc.subject.other	Mutant enzymes	en
dc.subject.other	pH changes	en
dc.subject.other	pH electrodes	en
dc.subject.other	Potentiometric assay	en
dc.subject.other	Reproducibility	en
dc.subject.other	Semi-permeable membranes	en
dc.subject.other	Site-directed mutagenesis	en
dc.subject.other	Substrate binding	en
dc.subject.other	Wild-type enzyme	en
dc.subject.other	Xenobiotic compounds	en
dc.subject.other	Xenobiotics	en
dc.subject.other	Enzyme inhibition	en
dc.subject.other	1 chloro 2,4 dinitrobenzene	en
dc.subject.other	glutathione transferase	en
dc.subject.other	malathion	en
dc.subject.other	xenobiotic agent	en
dc.subject.other	article	en
dc.subject.other	calibration	en
dc.subject.other	computer model	en
dc.subject.other	enzyme activity	en
dc.subject.other	enzyme structure	en
dc.subject.other	kinetics	en
dc.subject.other	pH	en
dc.subject.other	sensitivity analysis	en
dc.subject.other	Binding Sites	en
dc.subject.other	Biosensing Techniques	en
dc.subject.other	Enzyme Stability	en
dc.subject.other	Enzymes, Immobilized	en
dc.subject.other	Glutathione Transferase	en
dc.subject.other	Hydrogen-Ion Concentration	en
dc.subject.other	Malathion	en
dc.subject.other	Mutation	en
dc.subject.other	Potentiometry	en
dc.subject.other	Protein Engineering	en
dc.subject.other	Xenobiotics	en
dc.subject.other	Zea mays	en
dc.subject.other	Zea mays	en
dc.title	Engineering sensitive glutathione transferase for the detection of xenobiotics	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.bios.2008.06.037	en
heal.publicationDate	2008	en
heal.abstract	Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20 μM. The method's reproducibility was in the order of ±3-5% and malathion recoveries were 96.7 ± 2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme. © 2008 Elsevier B.V. All rights reserved.	en
heal.journalName	Biosensors and Bioelectronics	en
dc.identifier.issue	3	en
dc.identifier.volume	24	en
dc.identifier.doi	10.1016/j.bios.2008.06.037	en
dc.identifier.spage	498	en
dc.identifier.epage	503	en