The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in K M,cell app and 42% increase in V MAX, resulting in a 4-fold increase in k cat,cell/ K M,cell app. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K M,cell app, V MAX, k cat,cell, and k cat,cell/ K M,cell app.

A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione−ferricyanide double mediator system. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain.

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