ADH-NAD

Cyclic voltammetry was performed with the ADH-NAD-MB/polypyrrole electrode in 0.1 M phosphate buffer (pH 8.5) at a scan rate of 5 mV s l. The corresponding substrate of ADH caused the anodic current at +0.35 V vs. Ag/AgCl to increase. These results suggest a possible electron transfer from membrane-bound ADH to the electrode through membrane-bound NAD and MB with the help of the conductive polymer of polypyrrole. 

Rubianes and Rivas also proposed the immobilization of ADH (12.0 % w/w) into a CNTPE in the presence of NAD (12.0 % w/w) [79]. Based on the excellent electrocatalytic properties of MWCNTs towards the oxidation of NADH, a very fast and sensitive response for ethanol was obtained at CPE-ADH-NAD. ... 

Figure 39. The mechanism of ADH/NAD/MB-based biosensor response for ethanol ( Reprinteed from J. Electroanalytical Chemistry, 547, A.S. Santos, R.S. Freire, L.T. Kubota, Highly stable amperometric biosensor for ethanol based on Meldola s blue adsorbed on silica gel modified with niobium oxide, 137, Copyright(2003) with permission from Elsevier.

The substrate models concerned were fitted into the model of the active site of alcohol dehydrogenase-nicotinamide adenine dinucleotide (ADH-NAD) with VDW contacts, etc. not considered explicitly. 

Dutler and Branden (11, 12) have studied the interaction of the ADH-NAD complex with alkyl-substituted cyclohexanols to determine productive substrate orientations in the active site. Some of their findings were that 1) cyclohexanol fits best in a chair conformation with axial-reacting hydroxyl oxygen and equatorial-reacting hydrogen atoms on Cl, 2) that substitutions at Ck (para) have little effect on the reaction rate due to hydrophobic bonding with the residues in the hydrophobic "barrel region of the active site, 3) substitutions at C2,... 

The cofactor of ADH, NAD+, may not be replaced by other electron acceptors. Malinauskas and Kulys (1978) attempted to construct a reagentless alcohol sensor by coimmobilizing ADH with dextran-bound NAD+ by a dialysis membrane in front of an oxygen electrode. The O2 consumption was indicated via the reoxidation of NADH by NMP+. The system has also been used to measure NAD+ with high sensitivity. In... 

In a further study continuous electrolytic oxidation of ethanol was performed with the molecular-interfaced ADH/MB/NAD. The electrode potential was controlled at 0.35 V vs. Ag/AgCl. Ethanol in a solution was enzymatically oxidized to acetaldehyde with resulting in the reduction of NAD to NADH. The turnover number of the NAD/NADH cycle was calculated from the total conversion of ethanol and the coulomb during the electrolysis. The results clearly showed that NAD was electrochemically regenerated within the polypyrrole membrane through the electron transfer from ADH, NAD and MB to the electrode. 

ATP D-frnctose-6-phosphate 1-phosphotrans-ferase, and alcohol dehydrogenase (ADH), or alcohol NAD oxidoreduetase, which catalyze the reactions shown here. 

The leading substrate (A) is nicotinamide adenine dinucleotide (NAD ), and NAD and NADH (product Q) compete for a common site on E. A specific example is offered by alcohol dehydrogenase (ADH) ... 

Ethanol Electrodes The reliable sensing of ethanol is of great significance in various disciplines. The enzymatic reaction of ethanol with the cofactor nicotinamide-adenine dinucleotide (NAD+), in the presence of alcohol dehydrogenase (ADH)... 

Both ADH and ALDH use NAD+ as cofactor in the oxidation of ethanol to acetaldehyde. The rate of alcohol metabolism is determined not only by the amount of ADH and ALDH2 enzyme in tissue and by their functional characteristics, but also by the concentrations of the cofactors NAD+ and NADH and of ethanol and acetaldehyde in the cellular compartments (i.e., cytosol and mitochondria). Environmental influences on elimination rate can occur through changes in the redox ratio of NAD+/NADH and through changes in hepatic blood flow. The equilib-... 

FIGURE 12.10 Oxidation of methanol to C02, catalyzed by NAD+-dependent alcohol (ADH), aldehyde (AldDH), and formate (FDH) dehydrogenase, with regeneration of NAD+ via redox mediation to dia-phorase. (From [91], with permission from Elsevier.)... 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

The amperometric dehydrogenase sensor for ethanol consists of a platinum electrode on the surface of which alcohol dehydrogenase (ADH), Meldra blue (MB) and NAD are immobilized with a conductive polypyrrole membrane as schematically illustrated in Fig.24. 

A platinum disk electrode was electrolytically platinized in a platinum chloride solution to increase the surface area and enhance the adsorption power. The platinized platinum electrode was then immersed in a solution containing 10 mg ml l ADH. 0.75 mM and 6.2 mM NAD. After sufficient adsorption of these molecules on the electrode surface, the electrode was transferred into a solution containing 0.1 M pyrrole and 1 M KC1. Electrochemical polymerization of pyrrole was conducted at +0.7 V vs. Ag/AgCl. The electrolysis was stopped at a total charge of 1 C cm 2. An enzyme-entrapped polypyrrole membrane was deposited on the electrode surface. 

An electron transfer type of enzyme sensor was thus fabricated by a electrochemical process. Although no appreciable leakage of ADH and MB from the membrane matrix was detected, NAD leaked slightly. To prevent this leakage, the ADH-MB-NAD/polypyrrole electrode was coated with Nation. A calibration curve is presented in Fig.25 for ethanol determination in an aquous solution with the enzyme sensor. Ethanol is selectively and sensitively determined in the concentration range from 0.1 nM to 10 mM. 

In a further development, an ADH-MB-NAD/polypyrrole electrode, a platinum counter electrode and an Ag/AgCl reference electrode were assembled and covered with a gas-permeable polymer membrane to form an gaseous ethanol sensor. This appears to be the first time that a complete enzyme sensor for gaseous ethanol has been fabricated in such a manner with NAD incorporated in immobilized form. 

Fig. 6. Schematic representation for the ADH-catalyzed electroenzymatic oxidation of 2-hexene-l-ol and 2-butanol with indirect electrochemical NAD+ regeneration using (3,4,7,8-tetramethyl-l.lO-phenanthroline) iron(II/III) [Fe(tmphen)3] as redox catalyst...

ADH, alcohol dehydrogenase CYP, cytochrome P450 NAD, nicotinamide-adenme dinucleotide NADPH, nicotmamide-adenine dinucleotide phosphate (reduced form)... 

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