Oxidation using enzymes

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

Almost all types of cell can be used to convert an added compound into another compound, involving many forms of enzymatic reaction including dehydration, oxidation, hydroxyla-tion, animation, isomerisation, etc. These types of conversion have advantages over chemical processes in that the reaction can be very specific, and produced at moderate temperatures. Examples of transformations using enzymes include the production of steroids, conversion of antibiotics and prostaglandins. Industrial transformation requires the production of large quantities of enzyme, but the half-life of enzymes can be improved by immobilisation and extraction simplified by the use of whole cells. 

Meat products have to be stabilised in some cases, as meat lipids contain no natural antioxidants or only traces of tocopherols. Most muscle foods contain, however, an efficient multi-component antioxidant defence system based on enzymes, but the balance changes adversely on storage. The denaturation of muscle proteins is the main cause of the inbalance as iron may be released from its complexes, catalysing the lipid oxidation. Salting contributes to the negative effects of storage, as it enhances oxidation. Using encapsulated salt eliminates the deleterious effect of sodium chloride. 

There have been a number of reports of electrocatalysis of alcohol oxidation using immobilized PQQ-dependent alcohol dehydrogenases or flavin-containing alcohol dehydrogenases or oxidases with dissolved mediators in solution. Co-immobihzing the mediator with the enzyme is advantageous, as set out in Section 17.1, and several such strategies have been employed for electrocatalytic alcohol oxidation. 

Oxidised) organic molecules oxidation using iron but most notably copper zinc in transcription factors and hydrolytic enzymes (Chapter 8). 

The first enzyme biosensor was a glucose sensor reported by Clark in 1962 [194], This biosensor measured the product of glucose oxidation by GOD using an electrode which was a remarkable achievement even though the enzyme was not immobilized on the electrode. Updark and Hicks have developed an improved enzyme sensor using enzyme immobilization [194], The sensor combined the membrane-immobilized GOD with an oxygen electrode, and oxygen measurements were carried out before and after the enzyme reaction. Their report showed the importance of biomaterial immobilization to enhance the stability of a biosensor. 

Each reaction of p oxidation is catalyzed by a different enzyme. Chemically, they re pretty much the same as the reverse of the individual reaction of fatty acid synthesis, with two exceptions (1) p oxidation uses FAD for the formation of the double bond at the C-2 position, and (2) the reactions occur with the fatty acid attached to CoA rather than to the pantetheine of a multienzyme complex. 

TCF [Totally Chlorine-Free] A generic term for pulp-bleaching processes which do not use chlorine in any form. Oxidants and enzymes are used. See also ECF. 

Microorganisms, plants, and mammalian systems all contain enzymes capable of catalyzing chemical transformations with alkaloid substrates. Interesting and useful enzyme reactions that may occur with alkaloids include oxidations, reduc-... 

The enzyme p-ethylphenol methylene hydroxylase (EPMH), which is very similar to PCMH, can also be obtained from a special Pseudomonas putida strain. This enzyme catalyzes the oxidation of p-alkylphenols with alkyl chains from C2 to C8 to the optically active p-hydroxybenzylic alcohols. We used this enzyme in the same way as PCMH for continuous electroenzymatie oxidation of p-ethylphenol in the electrochemical enzyme membrane reactor with PEG-ferrocene 3 (MW 20 000) as high molecular weight water soluble mediator. During a five day experiment using a 16 mM concentration of p-ethylphenol, we obtained a turnover of the starting material of more than 90% to yield the (f )-l-(4 -hydroxyphenyl)ethanol with 93% optical purity and 99% enantiomeric excess (glc at a j -CD-phase) (Figure 14). The (S)-enantiomer was obtained by electroenzymatie oxidation using PCMH as production enzyme. 

Another useful enzyme combination is the tandem galactose oxidase— glycosidase. An example is the successive use of galactose oxidase from Dactylium dendroides and (S-A -acetylhexosaminidase from Talaromyces flavus, followed by in situ chemical oxidation, which afforded an immuno-active disaccharide of (3-D-GalpNAcA-(l — 4)-D-GlcpNAc76 (Scheme 3). 

In an article published independently at the same time, Stuehr and Ikeda-Saito89 used the purified bNOS and iNOS to reach the same conclusions. While the paper was under review, the authors mention that White and Marietta had reported earlier that the iron was a heme and was used as an oxidant. Using the same type of study, they found that the iron prophyrin and its CO derivative had the expected properties and proposed that the iron is penta-coordinated, with a cysteine thiolate as the fifth coordinate. A third publication confirmed the results when McMillan and coworkers90 used bNOS grown in human kidney cells. These workers obtained similar data for the light absorption of the enzyme and its CO spectrum. They also speculate on very similar sequences in the three types of purified enzyme that might be the porphyrin binding site. 

Similar to the AAOs, the aaDHs catalyze oxidative deamination, forming an oxoacid and ammonia. However, rather than using enzyme-hound FAD as the oxidant, followed hy O2, these enzymes employ nicotinamide cofactors, NAD or NADP, in free solution (Equation (3)). 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

Up to now the mediator properties have been discussed focusing on MET. There is however an additional aspect of the entire mediator concept. Mediators are also molecules that facilitate enzymatic oxidation of other substrates, the oxidation of which by enzymes only is slow (236,237). They are oxidative mediators that speed up the oxidation of poor substrates. They are oxidized by enzymes first and then, using their oxidative potential, react with substrate molecules to form product(s). Equations (47) and (48) illustrate the mechanism of action of oxidative mediators in catalysis by HRP. 

Several successful experiments using enzymes on electrodes have been conducted, although the problem of inactivation due to crash landings of the enzyme on the electrode during adsorption from solution is a hazard. 02 reduction and H2 oxidation have been successfully accelerated by enzyme-covered electrodes. 

Pyruvate ferredoxin oxidoreductase. Within Clostridia and other strict anaerobes this enzyme catalyzes reversible decarboxylation of pyruvate (Eq. 15-35). The oxidant used by clostridia is the low-potential iron-sulfur ferredoxin.320 3203 Clostridial ferredoxins contain two Fe-S clusters and are therefore two-electron oxidants. Ferredoxin substitutes for NAD+ in Eq. 15-33 but the Gibbs energy decrease is much less (-16.9 vs - 34.9 kj / mol. for oxidation by NAD+). 

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