Alcohol oxidase/dehydrogenase

NADH. Immobilized redox mediators, such as the phenoxazine Meldola Blue or phenothiazine compoimds, have been particularly useful for this purpose (20) (see also Figure 4-12). Such mediation should be useful for many other dehydrogenase-based biosensors. High sensitivity and speed are indicated from the flow-injection response of Figure 3-21. The challenges of NADH detection and the development of dehydrogenase biosensors have been reviewed (21). Alcohol biosensing can also be accomplished in the presence of alcohol oxidase, based on measurements of the liberated peroxide product. 

Weenen, H., Boog, R.L.G.M. and Apeldoorn, W. (1995) Aldehydes from alcohols using alcohol oxidases and dehydrogenases. Colloques - Institut National de la Recherche Agronomique, 75 (Bioflavour 95), 375-380. 

The work in the biosensor industry permitted the testing and proved of stability and reproducibility of enzymes, within the conditions employed in that area. Enzymes with demonstrated stability include lactate oxidase, malate dehydrogenase, alcohol oxidase, and glutamate oxidase. 

Alcohol oxidase was used to generate H202 followed by its reaction with luminol in the presence of K3[Fe(CN)6] as a catalyst [53], The luminescence was transmitted from the flow cell to the detector via optical fibers. Ethanol can be determined in the 3-750-pmol/L concentration range, with a detection limit of 3 pmol/L. Also, using an immobilized alcohol dehydrogenase reactor in glass beads, a FIA sensor for a reduced form of NADH was constructed by the ECL using the above-mentioned ruthenium tris(2,2 -biryridine) complex. The sensor was satisfactorily applied to the determination of ethanol concentration [54],... 

Alcohol is metabolized by alcohol dehydrogenase (a zero-order process except at very high and very low concentrations) to acetaldehyde, which is metabolize to carbon dioxide and water by aldehyde dehydrogenase. Catalase and the microsomal alcohol oxidase system are also involved. 

ACYL-CoA DEHYDROGENASES ADENYLYLSULEATE REDUCTASE ALCOHOL OXIDASE AMINE OXIDASES d-AMINO acid DEHYDROGENASE l-AMINO acid DEHYDROGENASE d-AMINO ACID OXIDASE l-AMINO ACID OXIDASE d-ASPARTATE OXIDASE BENZOATE 1,2-DIOXYGENASE... 

Poly(vinyl alcohol) oxidase PVA oxidase, poly(vinyl alcohol) oxidoreductase, poly(vinyl alcohol) dehydrogenase ... 

Different purified or partially purified enzymes were tested successfully, such as horse liver dehydrogenase [3], Sulfolobus solfataricus dehydrogenase [4], Pischia pastoris alcohol oxidase [5, 6], the baker s yeast alcohol dehydrogenase [7], and finally lipolytic enzymes, which probably constitute the major part of the work devoted to the use of enzymes working at the solid/gas interface, as summarized in a recent publication [8]. 

Q. Yao, S. Yabuki and F. Mizutani, Preparation of a carbon paste/alcohol dehydrogenase electrode using polyethylene glycol-modified enzyme and oil-soluble mediator, Sens. Actuators B Chem., 65(1-3) (2000) 147-149. A. Morales, F. Cespedes, E. Martinez-Fabregas and S. Alegret, Ethanol amperometric biosensor based on an alcohol oxidase-graphite-polymer biocomposite, Electrochim. Acta, 43(23) (1998) 3575-3579. 

Enzymes as different as yeast alcohol oxidase, mushroom polyphenol oxidase, and horse-liver alcohol dehydrogenase demonstrated vastly increased enzymatic activity in several different solvents upon an increase in the water content, which always remained below the solubility limit (Zaks, 1988b). While much less water was required for maximal activity in hydrophobic than in hydrophilic solvents, relative saturation seems to be most relevant to determining the level of catalytic activity. Correspondingly, miscibility of a solvent with water is not a decisive criterion upon transition from a monophasic to a biphasic solvent system, no significant change in activity level was observed (Narayan, 1993). Therefore, the level of water essential for activity depends more on the solvent than on the enzyme. 

Aldehydes are produced via the action of alcohol oxidizing enzymes. In those species studied, a primary alcohol oxidase which appears to require only molecular oxygen and the alcohol substrate is involved (5, 29, 33, 34). However, the action of alcohol dehydrogenases which do not require molecular oxygen to function can not be discounted where aldehydes or ketones are produced as pheromones by other insects. 

The alcohol dehydrogenases were already described in Chapter 3. These enzymes are cofactor dependent and in the active site hydrogen transfer takes place from NADH or NADPH. In the reverse way they can, however, be applied for the oxidation of alcohols in some cases (see below). Oxidases are very appealing for biocatalytic purposes, because they use oxygen as the only oxidant without the need for a cofactor. Oxidases usually have flavins (glucose oxidase, alcohol oxidase) or copper (examples galactose oxidase, laccase and tyrosinase) in the active site [18]. The mechanism for glucose oxidase (GOD) is denoted in... 

By using fluorescence techniques as little as 0.1 / g/ml of ethanol can be determined (59). A number of other lower aliphatic alcohols may be determined with alcohol dehydrogenase or with alcohol oxidases (60). 

Cyclopropanone hydrate forms a stable thiohemiketal with the active-site thiol which is not oxidized by nicotinamide adenine dinucleotide (NAD). This hydrate has also been found to be a suicide inhibitor for horseradish peroxidase flavoenzyme alcohol oxidase and quinoprotein alcohol dehydrogenases ... 

Acetaldehyde Ethanol Ethanol Glucose + O2 Alcohol dehydrogenase Candida utilis Alcohol oxidase... 

Over 90% of alcohol in the plasma is metabolized in the liver by three enzyme systems that operate within the hepatocyte. The remainder is excreted by the lungs and in urine and sweat. Alcohol is metabolized to acetaldehyde by alcohol dehydrogenase in the cell. In turn, acetaldehyde is metabolized to carbon dioxide and water by the enzyme aldehyde dehydrogenase. A second pathway for oxidation of alcohol uses catalase, an enzyme located in the peroxisomes and microsomes. The third enzyme system, the microsomal alcohol oxidase system, has a role in the oxidation of alcohol to acetaldehyde. These last two mechanisms are of lesser importance than the alcohol dehydrogenase-aldehyde dehydrogenase system. 

For enzymatic alcohol measurement, alcohol dehydrogenase (ADH, EC 1.1.3.13) and alcohol oxidase (AOD, EC 1.11.1.6) from various sources have been used ... 

Ethanol alcohol oxidase + alcohol dehydrogenase membrane O2 electrode Hopkins (1985)... 

Ethanol Alcohol oxidase + alcohol dehydrogenase Membrane Oj electrode [328]... 

Alcohol oxidase has been co-immobilized with alcohol dehydrogenase in order to increase the sensitivity of alcohol determination [328). In the presence of oxygen and NADH, ethanol is recycled between the two enzymes. A particular advantage of this system, which overcomes... 

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