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Amperometric determinations of reducing enzyme activities in living whole S. cerevisiae cells immobilized on micro-band electrodes

Jenny Emnéus 1,  Christer Spegel 5,  Arto Heiskanen 1,  Ted Johansson 2,  Marie-Francoise Gorwa-Grauslund 2,  Milena Koudelka 3,  Tautgirdas Ruzgas 4

1. Lund University, Department of Analytical Chemistry, Lund SE-221 00, Sweden
2. Lund University Department of Applied Microbiology, Box 124, Lund 22100, Sweden
3. Institute of Microtechnology, Rue Jaquet Droz 1, Neuchâtel 2007, Switzerland
4. Malmö Univerity Health and Society, None, Malmö 20506, Sweden
5. Sophion Bioscience, Baltorpvej 154, Ballerup 2750, Denmark

Abstract
A mediated amperometric method has been utilized for the determination of menadione (M) reducing enzyme activity as well as diketone reductase (DkR) activity in living, genetically modified, S. cerevisiae cells. The electrochemical results corresponded well to in vitro spectrophotometrically determined reductase activities. The amperometric method is based on M, a lipophilic quinone able to diffuse freely through the plasma membrane, and accept electrons from intracellular NAD(P)H dependent enzymes. The reduced form of M, menadiol, is extracellulary oxidised by ferricyanide, which in turn is oxidised at the platinum microband electrode. This process results in a current proportional to the activity of the intracellular M reducing enzymes [1].

It was found that the amplitude of the yeast-catalysed amperometric signal was dependent on the metabolic pathway supplying the NAD(P)H, in the order; ethanol oxidation pathway(EOP) > pentose phosphate pathway (PPP) > glycolytic pathway (GP). An optimum in the apparent electrochemical Michaelis-Menten (MM) constants for intracellular DkR was found for an M concentration of 200 mM, i.e. a minimum in the apparent KM and optimum in apparent imax.

[1] A. Heiskanen, J. Yakovleva, C. Spégel, R. Taboryski, M. Koudelka-Hep, J, Emnéus, T, Ruzgas, Electrochem. Com., 6 (2004) 219.

 

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Presentation: Poster at SMCBS'2005 Workshop, by Jenny Emnéus
See On-line Journal of SMCBS'2005 Workshop

Submitted: 2005-08-02 08:24
Revised:   2009-06-07 00:44