Dependent Dehydrogenase Enzymes Research Articles

Rapid Entrapment of Phenazine Ethosulfate within a Polyelectrolyte Complex on Electrodes for Efficient NAD+ Regeneration in Mediated NAD+-Dependent Bioelectrocatalysis.

Over the past two decades, the designs of redox polymers have become critical to the field of mediated bioelectrocatalysis and are used in commercial glucose biosensors, as well as other bioelectrochemical applications (e.g., energy harvesting). These polymers are specifically used to immobilize redox mediators on electrode surfaces, allowing for self-exchange-based conduction of electrons from enzymes far from the electrode to the electrode surface. However, the synthesis of redox polymers is challenging and results in large batch-to-batch variability. Herein, we report a rapid entrapment of mediators for NAD+-dependent bioelectrocatalysis within reverse ionically condensed polyelectrolytes. A high ionic strength aqueous solution of oppositely charged polyelectrolytes, composed of cationic polyguanidinium (PG) chloride and anionic sodium hexametaphosphate (P6), undergoes phase inversion into a solid microporous polyelectrolyte complex (PEC) when introduced into a low ionic strength aqueous solution. The ionic strength-triggered phase inversion of PGP6 solutions was investigated as a means to entrap mediators on the surface of electrodes for mediated bioelectrocatalysis. Compared to the traditional cross-linked immobilizations using redox polymers, this phase inversion takes place within seconds and requires up to 60 min for complete stabilization. In this work, redox mediator phenazine ethosulfate (PES) was entrapped within PGP6 on electrode surfaces for nicotinamide adenine dinucleotide (NAD+)-dependent bioelectrocatalysis. In the bulk solution, NAD+-dependent dehydrogenase enzymes catalyze the oxidation of the substrate while reducing NAD to reduced nicotinamide adenine dinucleotide (NADH). The resulting NADH is reoxidized to NAD+ by the entrapped PES that gets reduced on the electrode, completing the NAD+-regeneration-based bioelectrocatalysis. To show the use of these new materials in an application, biofuel cells were evaluated using four different anodic enzyme systems (alcohol dehydrogenase, lactate hydrogenase, glycerol dehydrogenase, and glucose dehydrogenase).

Quantum dots as nano plug-in's for efficient NADH resonance energy routing

The routing of fluorescent signals from NADH to quantum dots (QDs) has been a subject of extensive research for FRET based applications. In the present study, the spectral cross talk of NAD+/NADH with QDs was used to monitor the reaction of NAD+-dependent dehydrogenase enzyme. CdTe QD may undergo dipolar interaction with NADH as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, non-radiative energy transfer can take place from NADH to CdTe QD enhancing QDs fluorescence. Energy routing assay of NADH-QD was applied for detection of formaldehyde as a model analyte in the range 1000–0.01ng/mL by the proposed technique. We observed proportionate quenching of CdTe QD fluorescence by NAD+ and enhancement in the presence of NADH formed by various concentrations of enzyme (0.028–0.4U). Hence, it was possible to detect formaldehyde in the range 1000−0.01ng/mL with a limit of detection (LOD) at 0.01ng/mL and regression coefficient R2=0.9982. Therefore, a unique optical sensor was developed for the detection of the formaldehyde in sensitive level based on the above mechanism. This method can be used to follow the activity of NAD+-dependent enzymes and detection of dehydrogenases in general.

Enzymatic fuel cells: Integrating flow-through anode and air-breathing cathode into a membrane-less biofuel cell design

One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD +-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 μW cm −3 are reported, and characterization criteria that will aid in future optimization are discussed.

Simulation of Multistep Enzyme-Catalyzed Methanol Oxidation in Biofuel Cells

We report a simulation of multi-step enzyme catalysis and cofactor-mediated electron transfer in a bio-anode of a methanol biofuel cell. Three nicotinamide adenine dinucleotide (NAD+) dependent dehydrogenase enzymes, namely alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AlDH), and formate dehydrogenase (FDH) combine to achieve complete oxidation of methanol to carbon dioxide. Oxidation of methanol by the enzyme cascade produces the reduced co-factor, NADH, which is oxidized to regenerate NAD+ at an electrode comprised of a poly(methylene green) catalyst electrodeposited on carbon paper. Kinetic parameters for the enzymatic oxidation reactions were experimentally determined between pH 6.5 and 9 and were employed in the simulation to evaluate the effect of pH. Model results compare closely to previously reported experimental data over a broad pH range. Model-based analysis indicates that cell performance is controlled by NAD+ transport and NADH oxidation kinetics for electrode specific areas lower than 3 m2 cm−3 and shifted to enzyme kinetic control at higher specific area. An optimal electrode design is predicted to achieve current density exceeding 10 mA cm−2.

Electro-oxidative polymerization of phenothiazine dyes into a multilayer-containing carbon nanotube on a glassy carbon electrode for the sensitive and low-potential detection of NADH

An electrochemical sensing platform was developed for the amperometric detection of β-nicotinamide adenine dinucleotide (NADH) through the integration of a multi-walled carbon nanotube (MWCNT) into electropolymerized phenothiazine dyes. The composite containing MWCNT and poly(phenothiazine) was prepared by electro-oxidative polymerization of phenothiazine derivatives, Azure B, Azure A and thionine, into an MWCNT/ poly(diallyldimethylammonium chloride) (PDDA) multilayer, which was constructed by electrostatic layer-by-layer assembly on a glassy carbon electrode. The three phenothiazine monomers used in this study exhibited similar electrochemical behaviors. Azure B was used extensively as a model monomer for the investigation. Electrochemical techniques and scanning electron microscopy were used to demonstrate that the porous composite was formed and that the carbon nanotube served as a nano-sized backbone for the loading of polymeric phenothiazine. The electrocatalytic current for NADH oxidation was enhanced as the number of layers increased, implying that the increase of NADH-accessible poly(phenothiazine) and the three-dimensional arrangement of the poly(phenothiazine)-coated MWCNT in the composite facilitated electron and NADH transfer. Under optimal conditions, the detection limit for NADH decreases to 7.0 × 10−8 M at a potential of 0.1 V (versus Ag/AgCl) using a {MWCNT/PDDA}8−poly(Azure B) composite modified glassy carbon electrode, with a response time of about 5 s. This work demonstrates that the electropolymerization of the phenothiazine monomer into a pre-formed multilayer containing MWCNT can be used for the controllable preparation of stable MWCNT/poly(phenothiazine) composites on electrode surfaces, which have the potential to provide a platform for electrochemical biosensors based on NAD+-dependent dehydrogenase enzymes.

Modification of oxidative status in Plasmodium berghei-infected erythrocytes by E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline compared to chloroquine

E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline (IQ) is a new quinoline derivative which has been reported as a haemoglobin degradation and ss-haematin formation inhibitor. The haemoglobin proteolysis induced by Plasmodium parasites represents a source of amino acids and haeme, leading to oxidative stress in infected cells. In this paper, we evaluated oxidative status in Plasmodium berghei-infected erythrocytes in the presence of IQ using chloroquine (CQ) as a control. After haemolysis, superoxide dismutase (SOD), catalase, glutathione cycle and NADPH + H+-dependent dehydrogenase enzyme activities were investigated. Lipid peroxidation was also assayed to evaluate lipid damage. The results showed that the overall activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were significantly diminished by IQ (by 53.5% and 100%, respectively). Glutathione peroxidase activity was also lowered (31%) in conjunction with a higher GSSG/GSH ratio. As a compensatory response, overall SOD activity increased and lipid peroxidation decreased, protecting the cells from the haemolysis caused by the infection. CQ shared most of the effects showed by IQ; however it was able to inhibit the activity of isocitrate dehydrogenase and glutathione-S-transferase. In conclusion, IQ could be a candidate for further studies in malaria research interfering with the oxidative status in Plasmodium berghei infection.

Preparation of chitosan–dopamine-multiwalled carbon nanotubes nanocomposite for electrocatalytic oxidation and sensitive electroanalysis of NADH

A novel nanocomposite of glutaraldehyde crosslinked chitosan (CS)–dopamine (DA) composite with multiwalled carbon nanotubes (MWCNTs) was synthesized for low-potential and sensitive amperometric sensing of reduced nicotinamide adenine dinucleotide (NADH). The composite was characterized by FT-IR spectrophotometry. The nanocomposite modified Au electrode showed well-defined quinone/phenol redox peaks of the dopamine moieties and was able to effectively mediate the oxidation of NADH in pH 7.0 phosphate buffer, with an overpotential decrease by ca. 450 mV (vs. bare Au). The NADH electrochemical sensor showed a linear range of 0.1–600 μM, a limit of detection of 12 nmol L −1, good antiinterferent ability, and long-term stability. It was also found that the use of acidified MWCNTs showed better performance than that of the nonacidified MWCNTs. Both CS and DA being biomaterials, the CS–DA/MWCNTs system should be of interest and potential for developing in vivo NAD + (the oxidized form of NADH)-dependent dehydrogenase enzymes based biosensors.

Recent developments towards disposable screen-printed biosensors incorporating a carbon ink modified with the redox mediotor. Meldola's Blue

Screen-printed carbon electrodes (SPCEs) can be mass-produced at low cost and are readily adaptable as base transducers for incorporation into disposable biosensors. As biological recognition elements, the NAD + -dependent dehydrogenase enzymes have wide ranging application for the determination of many analytes in biological samples. In order to achieve the desired selectivity, it has proved necessary to reduce the overpotential required to electrochemically regenerate the cofactor, NAD + , by incorporating redox mediators into the carbon ink prior to screen-printing. One such electrocatalyst, Meldola's Blue (MB), has been used successfully and has been combined with dehydrogenase enzymes for the determination of some important biological analytes, including lactate, alcohol and ammonia. These examples from our laboratory using MB-modified SPCEs will be reviewed in this article, together with a recent application to determine aspartate aminotransferase (AST) in serum.

Amperometric biosensors based on NAD(P)‐dependent dehydrogenase enzymes

AbstractThis review deals with the use of pyridine‐nucleotide dependent dehydrogenase enzymes in amperometric biosensor design. The electrochemistry of the nicotinamide coenzymes, their mechanism and the challenges associated with their direct oxidation on solid electrodes are discussed. In addition, a survey of the different solutions proposed in the literature for addressing these problems and some of their applications are presented.

Evaluation of the factors influencing reactivity and stereospecificity in NAD(P)H dependent dehydrogenase enzymes

Calculations of AM1 potential energies for conformations of N-(1,β-ribosyl)-1,4-dihydronicotinamide (3H) and N-(1,β-ribosyl)nicotinamide (3), as defined by the nicotinamide ring deformation angles α C and α N and the torsion angles X n and X am , have been carried out. X n is the angle of rotation of the nicotinamide ring relative to the ribose ring and the conformers are designated as syn (nicotinamide-CONH 2 and ribose ring O are close) and anti (nicotinamide-CONH 2 and ribose ring O are distant). The angles α C and α N reflect the degree of bending of nicotinamide N and C4 out of the plane with C2, C3, C5, and C6 to provide quasi-boat and quasi-half-chair conformations

Colon carcinogenesis and the role of dehydrogenase enzyme activity: Inhibition of tumorigenesis by pyrazole

Dimethylhydrazine and the products of its metabolism, azoxymethane and methylazoxymethanol, are potent inducers of colon tumors in experimental animals. The marked sensitivity of the colon to these substances, as compared with the other segments of intestine, appears to be related to the presence of dehydrogenase enzymes in colonic epithelium able to activate methylazoxymethanol. Results of several studies indicate that the organotropic effects of this carcinogen are related to NAD +/NADP +-dependent dehydrogenase enzyme activity and that the product of this reaction, the aldehydic form of methylazoxymethanol, is the agent responsible for the observed biologic effects. Rats treated with pyrazole, an inhibitor of NAD +-dependent alcohol dehydrogenase, prior to their receiving methylazoxymethanol, are protected against the acute toxicity induced by this agent and do not develop any intestinal tumors. In related studies aimed at determining the mechanism of resistance to colon tumor induction in Lobund Wistar rats, it was found that this phenomenon is not due to differences in metabolic activation of carcinogen.