Ethanol enzymatic analysis

To measure ethanol in blood, enzymatic analysis is the method of choice for many laboratories. In this method, ethanol is measured by oxidation to acetaldehyde with NAD, a reaction catalyzed by alcohol dehydrogenase (ADH). With this reaction, formation of NADH, measured at 340nm, is proportional to the amount of ethanol in the specimen. The reaction is driven almost completely to the right by use of excess NAD and ADH and agents such as semicarbazide or tris (hydroxymethyl) aminomethane to trap acetaldehyde as it is formed. ... 

Barzana E, Klibanov AM, Karel M. 1989. A colorimetric method for the enzymatic analysis of gases The determination of ethanol and formaldehyde vapors using solid alcohol oxidase. Anal Biochem 182 109-115. 

The alcohol in a distillate can be estimated from its specific gravity or refractive index or more occasionally by chemical or enzymatic analysis. These last methods are more sensitive and are usually reserved for measuring small quantities of ethanol. For example, the alcohol in blood may be estimated by microdiffusion into a dilute acidified solution of potassium dichromate where it is oxidized to acetaldehyde while the orange-red dichromate is reduced to a green chromous salt ... 

Ukeda H., Kamikado H., Matsumoto K. and Osajima Y. (1989) A new approach of the coimmobilization of alcohol dehydrogenase and NAD on glutaraldehyde activated Sepharose and its application to the enzymatic analysis of ethanol. Agric. Biol. Chem., S3, 25-30. 

One hundred grams of fresh beets from five randomly selected roots are used for analysis. The tissue is blended for 1 min with 100 ml ethanol/water mixture (50 50, v/v) under a stream of nitrogen to lessen oxidative enzymatic reactions. The blender walls are washed with 100 ml of water, and the mixture is blended for an additional 5 min under nitrogen. Fifty grams of this homogenate are removed, filtered over a 10-g bed of celite, and washed with 300-400 ml of water until the tissue-celite mixture is colorless. The filtrate is quantitatively transferred to a 500-ml volumetric flash and brought to volume. An adequate dilution (1 5) of this sample is used for HPLC analysis. 

The analysis of TNT in wastewaters is made simple and direct by LC using a UV detector at 220nm (Refs 81 158). An LC method suitable for the low level determination of Tetrvl in the presence of TNT, RDX and HMX is described (Ref 91). The adsorptive LC of TNT was demonstrated using poly(styrene-divinyl benzene) adsorbent and ethanol as the moving phase (Ref 112). HPLC was used for the separation of TNT from purification by-products of hexanitro-bibenzyl (Ref 69). Enzymatic action on TNT was supported by HPLC (Ref 155). HPLC chromatograms of TNT are included, together with data on TLC and color reactions of TNT in mixts (Ref 153). Pollutants in wastewater... 

As a rule, the combined immobilization of oxidase and peroxidase on the electrode allows the determination of metabolite (S) concentration, even if negligibly small. This type of biosensor is the main one used for detection of any particular compound in blood or other multicomponent system glucose, ethanol, cholesterol, proteins, amino acids, etc. For example, for patients with diabetes a rapid analysis of their blood for glucose concentration is a vitally important procedure. We will not attempt to discuss in full all the existing types of biosensor we will just note that enzymatic and cell biosensors are the most widespread types of these appliances. Enzymatic biosensors are more appropriate to the subject of the current monograph and, therefore, they will be discussed below. 

The ultimate goal of the AFEX treatment is to increase the yields of fermentation products such as ethanol by increasing the digestibility of the biomass. Therefore, selected runs that showed higher glucan and xylan conversion were chosen for further SSF analysis. The best treatment temperature is to be selected based on the fermentation results, not just on enzymatic hydrolysis the fermentation results are presented later in this article. 

In spite of all these difficulties, microdialysis has been successftilly coupled to several analytical techniques for the bioprocess monitoring. HPLC is commonly used and such systems have been apphed in the analysis of oligosaccharides [44,180], ethanol [181], enzymatic digestion of lactose in milk [182], studies on drug dissolution [183], saccharides in wastewater [184], starch hydrolysis products [185]. 

Traditional methods of analysis for determination of principal organic acids, glycerol and sugars in wine are based on enzymatic or colorimetric reactions ethanol is determined by distillation of wine and density measurement of the distillate. In Table 1.2, data of organic acids, glycerol, glucose, fructose and ethanol determined by HPFC are reported as a percentage of results obtained by the traditional methods. 

As already indicated, a special problem with esters is their preparation from two natural precursor molecules by a chemical ester synthesis. Such products have to be labelled nature-identical. For an interesting positional H-NMR study on ethyl butyrate from enzymatic esterification of beet ethanol with butyric acid from milk see [317]. Another chance to detect a corresponding adulteration would be a positional carbon and oxygen isotope analysis of the ester components. Isotope effects on the esterification reaction in question seem to influence characteristically the 8-values of the atoms involved, and hence form a basis for the origin assignment of these compounds (for further details see 6.2.2.4.4). 

Torget R, Werdene P, Hinmel M, Grohman K (1990) Appl Biochem Biotechnol 24/25 115 Schell DJ, RUey C, Walter P, Bergeron P (1990) Technical and Economic Analysis of an Enzymatic Hydrolysis Based Ethanol Plant. National Renewable Energy Laboratory proprietary draft report... 

A. F. Gomez, J. R. Polonio, M. D. Luque de Castro, and M. Valcarcel Cases, Automatic Enzymatic-Fluorimetric Determination of Ethanol in Blood by Flow Injection Analysis. Anal. Chim. Acta, 148 (1985) 131. 

On the other hand, analysis of table 1 shows that the glucose derepressed mutant Candida molischiana 35M5N isolated by Janbon et al. [13] is able to produce large quantities of p-glucosidases with interesting properties. The enzyme has got a wide activity spectrum, an optimum pH of 3.5 and is very stable at low pH value (78% activity recovered after 145 h at pH 3.5, 30°C). The enzyme is also stable in the presence of ethanol. Thus, this enzyme was naturally chosen to develop an enzymatic system able to enhance the aromatic quality of wines. 

This colour change is the principle on which the breath analyser operates. Instrumental methods of breath alcohol analysis have recently been reviewed [24]. Enzymatic methods are usually based on alcohol dehydrogenase which catalyses the oxidation of ethanol to acetaldehyde ... 

Thiamine (vitamin Bj) occurs in foods in free and bound forms, the free form predominates in cereals and plants, whereas the pyrophosphate ester is the main form in animal products. Acid hydrolysis is required to release thiamine from the food matrix. Enzymatic hydrolysis is then needed to convert phosphate esters to thiamine. Prior to CE analysis it is necessary to clean up samples by using ethanol to precipitate protein and by passing through an ion-exchange resin. Thiamine has been determined in meat and milk samples using MEKC with ultraviolet (UV) detection at 254 nm, obtaining comparable sensitivity to that achieved by HPLC using an ion-pair reversed-phase column with postcolumn derivat-ization and fluorescence detection. 

Later, an enzymatic method based on oxidation of ethanol by alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD) followed by spectrophotometric analysis was reported. Current methods for detecting ethanol in body fluids are predominantly based on physicochemical techniques. A gas-liquid chromatographic (GLC) method is the most widespread because it is easy, rapid, and has high specificity and accuracy. Analytical methods used to determine alcohol in body fluids are siunma-rized in Table 1. 

Aliphatic alcohols are easily separated by liquid chromatography (LC) using a reversed-phase column. Unfortunately, LC does not have adequate sensitivity to alcohols, and its application to analysis of ethanol in body fluids has been hindered. Davis et al. developed a liquid chromatographic method coupled with an enzymatic method that generated NADH in a precolumn ADH-catalyzed oxidation of ethanol. Since this method is not selective for ethanol, it has only limited value for determining ethanol in body fluids. 

Aden A, Foust T. (2009). Technoeconomic analysis of the dilute sulfuric add and enzymatic hydrolysis process for the conversion of com stover to ethanol. Cellulose, 16(4), 535—545. 

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