Enzymatic oxidation, ethanol

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

In order to assess the synthetic potential of enzymatic oxidations for organosilicon chemistry, the (hydroxyalkyl)silanes 95, 97 and 99 have been studied for their oxidation (dehydrogenation) with horse liver alcohol dehydrogenase (HLADH E.C. l.l.l.l)79. For this purpose, these compounds were incubated with HLADH in a TRIS-HC1 buffer/THF system in the presence of NAD+. As monitored spectrophotometrically (increase of absorbance of the NADH formed), the (2-hydroxyethyl)silane 97 and the (3-hydroxypropyl)silane 99 were better substrates for HLADH than ethanol, whereas the related (hydroxymethyl)silane 95 was not a substrate under the experimental conditions used. Interestingly, the corresponding carbon analogue 101 was found to be accepted by HLADH. On the other hand, the (2-hydroxyethyl)silane 97 was found to be a better... 

Of course, in the enzymatic oxidation of unlabeled ethanol one cannot operationally discern that the hydrogen abstracted is pro-R. Reactions of this type which are prostereoselective have been called stereochemically cryptic m>. 

Fig, 3,8 Enzymatic oxidation of ethanol to acetaldehyde [40]. ADH = alcohol dehydrogenase, NAD+ and FMN = cofactors, catalase = second enzyme to regenerate cofactors... 

Figure 4. Enzymatic oxidation of ethanol to acetaldehyde according to U.S. Patent 4 481 292.

X 10 M OP, with ADH for 1 hour at 0° C. at the indicated pH levels. OP completely inhibits the enzymatic oxidation of ethanol at pH values below 7.5, and inhibition diminishes with increasing alkalinity of the incubation medium. At pH levels below 6.0 and above 9.5, the activity of the uninhibited control reaction became too low and erratic to measure. Spectrophotometric data on the effect of pH on the formation of [Zn(OP) ]++ complexes from the work of McClure and Banks (1951) are shown for comparison. With rising pH, the formation of these complexes is progressively suppressed (Fig. 5). The enzymatic data plotted in Fig. 5 imply that the inhibition observed is a function of the formation of a zinc-OP complex. The dependence of the effectiveness of other inhibitors (vide supra) on pH of the preincubation mixture has been studied (Vallee and Hoch, in preparation for publication). 

PROBLEM 15.12 The mechanism of enzymatic oxidation has been studied by isotopic labeling with the aid of deuterated derivatives of ethanol. Specify the number of deuterium atoms that you would expect to find attached to the dihydropyridine ring of the reduced form of the nicotinamide adenine dinucleotide coenzyme following enzymatic oxidation of each of the alcohols given ... 

Barzana E, Karel M, KUbanov A (1989) Enzymatic oxidation of ethanol in the gaseous phase. Biotechnol Bioeng 34 1178-1185... 

In the presence of glucose, the PL quenching of the 02-sensitive dye is reduced (i.e., I and x increase) due to O2 consumption during the enzymatic oxidation/ A similar reaction in the presence of lactate oxidase (LOx) or alcohol oxidase (AOx) describes lactate or ethanol oxidation, respectively. 

Enzymatic oxidation of ethanol and butanol Alcohol oxidase Toluene Conversion increases with increasing volume fraction of organic phase Hidaka et al. (1995)... 

Acetaldehyde is mostly formed by the enzymatic oxidation of ethanol, the latter is formed in roots under anoxic conditions or in anoxic stems of woody plants [13]. Large fluxes of acetaldehyde have been observed from the leaves when roots are flooded (limited access to oxygen) or exposed to anoxia. It has also recently been discovered that acetaldehyde can arise in leaves directly from metabolism that occurs during light-dark transitions [14]. 

The physical processes of racking and clarification (with bentonite) help remove the enzyme fraction associated with particulate materials (Gortegs and Geisenheim, 1986). In addition, the activity of tyrosinase decreases during yeast fermentation, largely because of the production of ethanol. Consequently, clarified protein-stable wines made from relatively sound berries are unlikely to contain significant quantities of either laccase or tyrosinase and, hence, should undergo little enzymatic oxidation (Simpson, 1980). 

Pyridone of N -methylnicotinamide forms white crystals from acetone with a molecular weight of 152.15 it melts at 212 214° and is soluble in water and ethanol. It may be prepared from coumalic acid by ring closure with methyl amine. It is the sole product of the enzymatic oxidation of N -methylnicotinamide with a quinine-oxidizing enzyme, and is also obtained from A i-methylnicotinamide or the AT i-methylbetaine of nicotinic acid by oxidation with alkaline ferricyanide and subsequent treatment with SOCla and NHa. 

Several other sensors are available that arc based on the voltammetric measurement of hydrogen peroxide produced by enzymatic oxidations of other species of clinical interest. These analytes include sucrose, lactose, ethanol, and (.-lactate. A (Afferent enzyme is. of course, required for each species. In some cases, enzyme electrodes can be based on measuring oxygen or on measuring pH as discussed in Section 23F-2. 

Special preparation and extraction techniques have been described such as DHAA reduction, HPOs/ethanol utilization, enzymatic oxidation and o-phenUendiamine... 

Acetic acid is formed through the enzymatic oxidation of ethanol produced by fermentation. Vinegar is the term given to the dilute (ca. 4-12%) aqueous solution thus generated in ciders, wines, and malt extracts. Louis Pasteur in 1864 established the involvement of bacteria in the oxidation stage of this ancient process. 

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. 

The microsomal ethanol oxidizing system is another mechanism of ethanol metabolism. CYP2E1 may be an important enzyme in the metabolism of ethanol in heavy drinkers, who may have a 10-fold increase in activity. Two aUehc variants in the gene cl and c2) are associated with differing enzymatic activity. Approximately 40% of Japanese have the more active c2 allele, which is rare in individuals of European heritage (Sun et al. 2002). It is not believed to be a risk or protective factor in the development of alcohohsm, although current studies are examining its relationship to a variety of ethanol-related diseases. 

Frimmer U, F Widdel (1989) Oxidation of ethanol by methanogenic bacteria. Growth experiments and enzymatic studies. Arch Microbiol 152 479-483. 

Recent studies suggest that many factors may affect hydroxyl radical generation by microsomes. Reinke et al. [34] demonstrated that the hydroxyl radical-mediated oxidation of ethanol in rat liver microsomes depended on phosphate or Tris buffer. Cytochrome bs can also participate in the microsomal production of hydroxyl radicals catalyzed by NADH-cytochrome bs reductase [35,36]. Considering the numerous demonstrations of hydroxyl radical formation in microsomes, it becomes obvious that this is not a genuine enzymatic process because it depends on the presence or absence of free iron. Consequently, in vitro experiments in buffers containing iron ions can significantly differ from real biological systems. 

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