Electrochemical Probing of in Vivo 5-Hydroxymethyl Furfural Reduction in Saccharomyces cerevisiae

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• 作者：Natalie V. Kostesha ; Joo R. M. Almeida ; Arto R. Heiskanen ; Marie F. Gorwa-Grauslund ; Barbel Hahn-Hgerdal ; Jenny Emnus
• 刊名：Analytical Chemistry
• 出版年：2009
• 出版时间：December 15, 2009
• 年：2009
• 卷：81
• 期：24
• 页码：9896-9901
• 全文大小：178K
• 年卷期：v.81,no.24(December 15, 2009)
• ISSN：1520-6882

文摘

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. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. 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. 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 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. 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. These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic parameters in S. cerevisiae cells.