• Journal of Microbiology
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• v.43 no.5
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• pp.451-455
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• 2005

In this study, whole cells and a crude enzyme of Candida peltata were applied to an electrochemical bioreactor, in order to induce an increment of the reduction of xylose to xylitol. Neutral red was utilized as an electron mediator in the whole cell reactor, and a graphite-Mn(IV) electrode was used as a catalyst in the enzyme reactor in order to induce the electrochemical reduction of $NAD^+$ to NADH. The efficiency with which xylose was converted to xylitol in the electrochemical bioreactor was five times higher than that in the conventional bioreactor, when whole cells were employed as a biocatalyst. Meanwhile, the xylose to xylitol reduction efficiency in the enzyme reactor using the graphite-Mn (IV) electrode and $NAD^+$ was twice as high as that observed in the conventional bioreactor which utilized NADH as a reducing power. In order to use the graphite-Mn(IV) electrode as a catalyst for the reduction of $NAD^+$ to NADH, a bioelectrocatalyst was engineered, namely, oxidoreductase (e.g. xylose reductase). $NAD^+$ can function in this biotransformation procedure without any electron mediator or a second oxidoreductase for $NAD^+/NADH$ recycling

• BMB Reports
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• v.28 no.5
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• pp.392-397
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• 1995

The effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the intracellular $Ca^{2+}$ level was studied in NIH3T3 fibroblast cells. A reduction of the intracellular $Ca^{2+}$ level was observed after exposure to 300 ${\mu}m$ MNNG. However, the intracellular level of $IP_3$, a well-known regulator of $Ca^{2+}$ release from internal storage, was not changed by MNNG treatment. Instead, a reduction of the intracellular NAD level was observed. NAD as well as $IP_3$ stimulated intracellular $Ca^{2+}$ release from permeabilized cells. The treatment of 3-aminobenzamide, which inhibited the MNNG-induced reduction of the NAD level, also prevented the MNNG-induced decrease of the $Ca^{2+}$ level. Our data suggest that MNNG reduces the intracellular $Ca^{2+}$ level by NAD depletion in NIH3T3 cells.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.10a
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• pp.181-188
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• 2000

The electrocatalytic reduction of NAD$^{＋}$ using diaphorase was studied. methyl viologen (MV$^{2＋}$) mediator between an electrode and the enzyme. Steady-state currents could be obtained under the conditions of slow scan rate, low MV$^{2＋}$concentration, and high NAD$^{＋}$ concentration as the electrode reaction was converted to an electrochemical-catalytic (EC') reaction. The biomecular rate constant for the reaction of the reduced methyl viologen with the oxidized diaphorase was estimated as 7.5$\times$10$^3$M$^{-1}$ s$^{-1}$ from the slope of the current versus [MV$^{2＋}$] plot. And the optimal concentrations of diaphorase, MV$^{2＋}$ and NAD$^{＋}$ in the mediated electrocatalytic reduction of NAD$^{＋}$ were decided by applying the cyclic voltammetry. The optimal concentrations of the species were obtained by finding the conditions which gave the highest and steady-state current at a gold-amalgam electrode. The highest and steady-state catalytic current was achieved under the conditions of 1.5 U/ml diaphorase, 0.2 mM MV$^{2＋}$, and 4.8 mM NAD$^{＋}$ at the scan rate of 2 mV s$^{-1}$ , suggesting that the rate of the electrocatalytic reation is the higest under the former conditions. The electrochemical procedure under the conditions of 1.5 U/ml diaphorase,0.2 mM MV$^{2＋}$, and 4.8 mM NAD$^{＋}$ was used favorably to drive an enzymatic reduction of pyruvate to D-lactate.

• BMB Reports
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• v.32 no.4
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• pp.321-325
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• 1999

NAD(P)H-quinone oxidoreductase (EC 1. 6. 99. 2) was purified form S. cerevisiae. The enzyme readily reduced 2,6-dichlorophenolindophenol, a quinonoid redox dye, as well as substituted benzo- and naphthoquinones, and could accept electrons from either NADH or NADPH. The purified NAD(P)H-quinone oxidoreductase turned out to be capable of reducing nitrosoarenes as well as a variety of quinones. A chemical-trapping technique using 4-chloro-1-naphthol was used to show that the N,N-dimethyl-p-benzoquinonediiminium cation was produced in the reduction of 4-nitroso-N,N-dimethylaniline catalyzed by NAD(P)H-quinone oxidoreductase.

• Bulletin of the Korean Chemical Society
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• v.22 no.11
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• pp.1192-1196
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• 2001

A diaphorase enzyme electrode for the catalytic reduction of NAD+ , the oxidized form of nicotinamide adenine dinucleotide, has been prepared. The enzyme layer grew spontaneously over an aminoethanethiol self assembled monolayer on a go ld plate electrode. The growth was accomplished by simply dipping the electrode covered by the aminoethanethiol monolayer into a solution containing both glutaraldehyde and diaphorase. We suggested that the glutaraldehyde as a cross-linking reagent was attached to the amino groups of the aminoethanethiol monolayer and the diaphorase enzyme molecules were bound to free aldehyde groups of the glutaraldehyde. Further attachments of the enzyme molecules over the bound enzyme molecules continued with the bridging of the glutaraldehyde. In frequency measurements with a quartz crystal microbalance, the frequency decrease was much more than it was for that of the enzyme monolayer formation, and an enzyme layer thicker than a monolayer was formed. The modified electrode was employed to reduce NAD+ , using diffusional methyl viologen as an electron transfer mediator. The NAD+ was electrocatalytically reduced, and the catalytic current was almost equivalent to that with the multilayered electrode of ten enzyme layers.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.914-918
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• 2004

This study was carried out to analyze the metabolic flux of W. kimchii sk10 on pyruvate and ethanol as a carbon source. The sk10 grown on ethanol produced acetate under aerobic conditions rather than under anaerobic conditions. The lactate and acetate were produced on ethanol plus pyruvate by the sk10 grown under aerobic and anaerobic conditions, respectively. The resting cell of sk10 produced 99.1 mM acetate and 17.3 mM lactate under aerobic conditions and 51.1 mM acetate and 62.4 mM lactate under anaerobic conditions from ethanol plus pyruvate, respectively. This result is thought to be due to the difference in the $NADH/NAD^+$ ratio depending on the growth conditions. The 11-fold overproduction of NADH peroxidase results in a low $NADH/NAD^+$ratio under aerobic growth conditions. At the low $NADH/NAD^+$ ratio, the metabolic flux of pyruvate toward lactate has to be shifted to a flux toward acetate without NADH oxidation to $NAD^+$, and ethanol oxidation to acetate coupled to $NAD^+$ reduction to NADH has to be activated.

• Journal of Life Science
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• v.24 no.1
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• pp.98-103
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• 2014

NAD(P)H quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes the two electron reduction of diverse substrates, including quinones. It uses NADH or NADPH as a cofactor for enzymatic machinery. In the metabolism of quinones, NQO1 has two conflicting functions because of the different stability of converted hydroquinones. The stable form of hydroquinone is excreted from cells by conjugation with glutathione or glucuronic acid. The unstable form of hydroquinone induces cell death by induction of oxidative stress and DNA damage. Certain quinones known as bio-reductive agents have a cytotoxic function following reduction by NQO1. Bio-reductive agents, such as ${\beta}$-lapachone or mitomycin C, induce the depletion of NAD(P)H and the generation of oxidative stress in an NQO1-dependent manner. NQO1 is highly expressed in several cancer tissues. Therefore, NQO1 is a good therapeutic target for cancer treatment with bio-reductive agents.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.540-546
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• 2004

We deviced a new graphite-Mn(II) electrode and found that the modified electrode with Mn(II) can catalyze NADH oxidation and $NAD^+$ reduction coupled to electricity production and consumption as oxidizing agent and reducing power, respectively. In fuel cell with graphite-Mn(II) anode and graphite-Fe(III) cathode, the electricity of 1.5 coulomb (A x s) was produced from NADH which was electrochemically reduced by the graphite-Mn(II) electrode. When the initial concentrations of pyruvate and acetaldehyde were adjusted to 40 mM and 200 mM, respectively, about 25 mM lactate and 35 mM ethanol were produced from 40 mM pyruvate and 200 mM acetaldehyde, respectively, by catalysis of ADH and LDH in the electrochemical reactor with $NAD^+$ as cofactor and electricity as reducing power. By using this new electrode with catalytic function, the bioelectrocatalysts are engineered; namely, oxidoreductase (e.g., lactate dehydrogenase) and $NAD^+$ can function for biotransformation without electron mediator and second oxidoreductase for $NAD^+$/NADH recycling.

• Journal of Electrochemical Science and Technology
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• v.2 no.3
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• pp.163-167
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• 2011

Nicotinamide adenine dinucleotide, $NAD^+$, and its reduced form, NADH, play important roles as coenzymes in many enzymatic reactions. Electrochemical methods for $NAD^+$ or NADH detection or generation are drawn attention because it can provide the simple and low cost platform with fairly good sensitivity. In this study, the polysiloxane viologen polymer/diaphorase/hydrophilic polyurethane (PSV/DI/HPU) modified electrodes were simply prepared and demonstrated for bio-electrocatalytic $NAD^+$ sensors. The electrodes were co-immobilized with diaphorase and polysiloxane viologen polymer as an electron mediator followed by the overcoating with HPU membrane. The mixture of the enzyme and the electron mediator was well stabilized within HPU membrane and exhibited good reversibility and stability. The sensitivity was 0.2 $nA{\cdot}{\mu}M^{-1}$ and the detection limit was 28 ${\mu}M$ with a response time of 50 s ($t_{90%}$). The capability for NADH sensor was also observed on the PSV/DI/HPU electrode.

• Applied Biological Chemistry
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• v.41 no.4
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• pp.228-234
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• 1998

An adaptive measure of photosynthetic cells to a condition identified with a reduction of cellular energy charge, caused by water deficit-induced impairment of photosynthetic ATP production, was investigated using hydroponically cultured rice seedlings. Water stress treatment of the seedlings resulted in a marked decrease in cellular ATP level, a significant increase in the content of NAD(H) and concurrent decrease in that of NADP(H) in shoots, which accompanied a decrease in the activity of NAD kinase (EC 2.7.1.23) that specifically converts NAD(H) to NADP(H). The decline in the enzyme activity was particularly evident in the $Ca^{2+}/calmodulin-dependent$ kinase, the major form of NAD kinase in plants, whereas the level of active calmodulin remained unchanged during water deficit. The ratio of $NADP^+$ to NADPH was maintained nearly constant and no increases were seen in the level of $H_2O_2$ and the activities of $superoxide/H_2O_2-detoxifying$ enzymes in shoots stress-treated for two days. Based on these results, it may be suggested that rice plants take a strategy to cope with an adverse situation of limited photophosphorylation created by water deficit in that cells facilitate ATP production through glycolysis and oxidative phosphorylation; in doing so, rice cells suppress NAD kinase activity, consequently up-sizing the NAD(H) pool at the expense of the NADP(H) pool. Several parameters associated with the stress symptoms are also of implicative that there is no overproduction of superoxide radical or the related active oxygen at least in rice seedlings.