title: An efficient amperometric transketolase assay: towards inhibitor screening.
abstract: This paper describes an innovative amperometric biosensor for the in vitro determination of activity of transketolase from Escherichia coli (TKec) using commercially available TK substrates, namely d-fructose-6-phosphate a physiological donor and glycolaldehyde the best non-phosphorylated acceptor. A galactose oxidase (GAOx) biosensor, based on the immobilization of this enzyme within laponite clay, allows amperometric detection of L-erythrulose released upon TK-catalyzed reaction. A calibration curve has been established from 0.01 to 0.1 U ml(-1) TKec concentration in solution. These data are comparable to that obtained by a fluorometric method. In order to ensure a higher sensitivity and re-usability of the system, an original bienzymatic sensing system was further developed based on apoenzyme TKec and GAOx separately immobilized on the electrode surface. The inner sensing layer contains GAOx@laponite and the outer layer TKec@layered double hydroxide biohybrid. The biosensor response was validated by the determination of KD(app) for thiamine diphosphate, the TK cofactor and the inhibition action of two commercially available products, pyrophosphate, a TK cofactor analog and d-arabinose-5-phosphate, a substrate analog.
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, UMR-CNRS 6296, F-63000 Clermont-Ferrand, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Touisni
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, UMR-CNRS 6296, F-63000 Clermont-Ferrand, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Charmantray
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, UMR-CNRS 6296, F-63000 Clermont-Ferrand, France.
authors.forename: Virgil
authors.initials: V
authors.lastname: Hélaine
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, UMR-CNRS 6296, F-63000 Clermont-Ferrand, France. Electronic address: Laurence.Hecquet@univ-bpclermont.fr.
authors.forename: Laurence
authors.initials: L
authors.lastname: Hecquet
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, UMR-CNRS 6296, F-63000 Clermont-Ferrand, France. Electronic address: Christine.Mousty@univ-bpclermont.fr.
authors.forename: Christine
authors.initials: C
authors.lastname: Mousty
mesh_terms: D001203:Ascomycota / Q000201:enzymology; D015374:Biosensing Techniques / Q000295:instrumentation; D055664:Electrochemical Techniques / Q000295:instrumentation*; D057075:Enzyme Assays / Q000295:instrumentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism; D004867:Equipment Design; D004926:Escherichia coli / Q000201:enzymology*; D005692:Galactose Oxidase / Q000378:metabolism; D017640:Silicates / Q000737:chemistry; D014174:Transketolase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D017640:Silicates; C524813:laponite; D005692:Galactose Oxidase; D014174:Transketolase
keywords: Amperometric biosensor; Clay modified electrode; Galactose oxidase; Inhibition; Layered double hydroxides.; Transketolase