NAD H

Biological activity (BA) was chosen as such parameter. The BA determined using a system and a technique for a class of natural polyphenolic bonds nicotinamide adenine dinucleotide restored (NAD H ) - ferricyanide (KjFe(CN)g) in a phosphates buffer solution. 

For the majority of redox enzymes, nicotinamide adenine dinucleotide [NAD(H)j and its respective phosphate [NADP(H)] are required. These cofactors are prohibitively expensive if used in stoichiometric amounts. Since it is only the oxidation state of the cofactor that changes during the reaction, it may be regenerated in situ by using a second redox reaction to allow it to re-enter the reaction cycle. Usually in the heterotrophic organism-catalyzed reduction, formate, glucose, and simple alcohols such as ethanol and 2-propanol are used to transform the... 

Wichmann, R., Wandrey, C., Biickmann, A.F. and Kula, M.-R. (1981) Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotechnology and Bioengineering, 23, 2789-2796. 

Fig. 21.1 The interactions between the bound coenzyme molecule and the amino acids at positions 47 and 369 in the / , / 2, and / 3 polymorphic variants as observed in their respective structures determined by X-ray crystallography. The dashed lines indicate possible hydrogen-bonds between the amino acids and the phosphate oxygens of the bound coenzyme molecule, NAD(H). Arg47 is substituted by a His residue in the f 2 isozyme and Arg369 is substituted by a Cys residue in the / 3 isozyme. In each case, the substitution results in a net loss of hydrogen-bonding interactions and weaker affinity for the coenzyme.

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

The most important coenzymes in synthetic organic chemistry [14] and industrially applied biotransformations [15] are the nicotinamide cofactors NAD/ H (3a/8a, Scheme 43.1) and NAD(P)/H (3b/8b, Scheme 43.1). These pyridine nucleotides are essential components of the cell [16]. In all the reactions where they are involved, they serve solely as hydride donors or acceptors. The oxidized and reduced form of the molecules are shown in Scheme 43.1, the redox reaction taking place at the C-4 atom of the nicotinamide moiety. 

NAD(H) from niacin (some may be synthesized from tryptophan)... 

Based on the experience mentioned previously, the Dimroth rearrangement was succesfully applied for the preparation of water-soluble macro-molecular adenosine derivatives of the redox enzymes 72 (NAD(H), NADP(H), and FAD) (Scheme IV.31) (86MI1 87MI3, 87MI2 88H1623 89MI1 90M11). 

Tricarboxylic acid cycle inhibited due to high ratio of NAD H/NAD ... 

L-Iactate is oxidized by lactate oxidase to pyruvate, which is reduced back to lactate by LDH. The total enthalpy change for this system can be further increased by addition of catalase, which makes the overall enthalpy change as large as -225 kJ/mol, so signal increases greater than 1000-fold can be obtained as a result. Co-enzyme recycling was also used for the determinations of ATP/ADP [161] and NAD(H) [162],... 

Wandrey, C. and Bossow, B. (1986) Continuous cofactor regeneration— utilization of polymer bound NAD(H) for the production of optically active acids. Biotechnol... 

The thermodynamics of the reaction are favorable if a large excess of ammonium is used. In a batch reactor the number of regenerations of NADH may be very high, but then the reaction should be started with a very small amount of it and the reaction rate would be very low. A sufficiently high rate is obtained if the concentration of NAD(H) is around its K value, but at those concentrations it will be necessary to recover it after the reaction. 

A solution to this problem is the enzyme membrane reactor (Figure 10.8). This is a kind of CSTR (continuous stirred tank reactor), with retains the enzyme and the cofactor using an ultrafiltration membrane. This membrane has a cut-off of about 10000. Enzymes usually have a molecular mass of 25000-250000, but the molecular mass of NAD(H) is much too low for retention. Therefore it is first derivatized with polyethylene glycol (PEG 20000). The reactivity of NAD(H) is hardly affected by the derivatization with this soluble polymer. Alanine can now be produced continuously by high concentrations of both enzymes and of NAD (H) in this reactor. 

In a photometric assay NADP(H)-dependent LBADH (see above) [9] and NAD(H) -dependent Candida parapsilosis carbonyl reductase (CPCR) [40] were identified as suitable catalysts accepting a broad range of ynones as substrates. Both enzymes catalyze the reduction of various aryl alkynones 21 with high enantioselectivity and efficiency (Scheme 2.2.7.13) [41]. 

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