Effect of yeast pretreatment on the characteristics of yeast-modified electrodes as mediated amperometric biosensors for lactic acid.
ABSTRACT Carbon paste electrode modified with baker' and wine yeast Saccharomyces cerevisiae (a source of flavocytochrome b(2)) were investigated as amperometric biosensors for L-lactic acid. Before immobilization on the electrode surface, yeast cells were pretreated with various electrolytes, alcohols and weak organic acids. Electrode responses to L-lactic acid were tested in the presence of various mediators (potassium ferricyanide, phenazine methosulfate, 2,6-dichlorophenolindophenol sodium salt hydrate, 1,2-naphthoquinone-4-sulfonic acid sodium salt). The highest (144+/-7 nA per 0.2 mM L-lactic acid) and the most stable responses were obtained after yeast pretreatment with 30% ethanol using potassium ferricyanide as a mediator. Different electrode sensitivities with mediator phenazine methosulphate probably reflected diverse changes in yeast membrane (and/or cell wall).
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ABSTRACT: In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 220.127.116.11, also known as flavocytochrome b(2), FC b(2)) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b(2) the O(2) reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b(2), cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN(-), lactate respiration could be proven for causing the decrease in the O(2) reduction.Bioelectrochemistry (Amsterdam, Netherlands) 05/2009; 76(1-2):175-9. · 2.65 Impact Factor
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ABSTRACT: This work describes a mediated amperometric method for simultaneous real-time probing of the NAD(P)H availability in two different phenotypes, fermentative and respiratory, of the phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44 [ENY.WA-1A pgi1-1D::URA3], and its parental strain, ENY.WA-1A. The developed method is based on multichannel detection using microelectrode arrays. Its versatility was demonstrated by using four microelectrode arrays for simultaneously monitoring the NAD(P)H availability of both geno- and phenotypes under the influence of two different carbon sources, glucose and fructose, as well as the cytosolic and mitochondrial inhibitor and uncoupler, dicoumarol. The obtained results indicate that the method is capable of accurately and reproducibly (overall relative standard error of mean 3.2%) mapping the real-time responses of the cells with different genotype-phenotype combinations. The ENY.WA cells showed the same response to glucose and fructose when dicoumarol was used; fermentative cells indicated the presence of cytosolic inhibition and respiratory cells a net effect of mitochondrial uncoupling. EBY44 cells showed cytosolic inhibition with the exception of respiratory cells when fructose was used as carbon source.Bioelectrochemistry (Amsterdam, Netherlands) 04/2009; 76(1-2):180-8. · 2.65 Impact Factor