NAD+ regeneration

A lot of analytical techniques have been proposed in recent decades and most of them are based on enzymes, called dehydrogenases, which are not sensitive to oxygen and need cofactors such as NAD". The key problems which seriously hamper a wide commercialization of biosensors and enzymatic kits based on NAD-dependent enzymes are necessity to add exogenous cofactor (NAD" ) into the samples to be analyzed to incorporate into the biologically active membrane of sensors covalently bounded NAD" to supply the analytical technique by NAD -regeneration systems. 

Fig. 4. Electroenzymatic oxidation of / -D-glucose with direct electrochemical NAD+ regeneration (GDH = glucose dehydrogenase)...

Fig. 6. Schematic representation for the ADH-catalyzed electroenzymatic oxidation of 2-hexene-l-ol and 2-butanol with indirect electrochemical NAD+ regeneration using (3,4,7,8-tetramethyl-l.lO-phenanthroline) iron(II/III) [Fe(tmphen)3] as redox catalyst...

Fig. 9. Schematic representation for the HLADH-catalysed enzymatic oxidation of cydohexanoi with indirect electrochemical NAD+ regeneration...

Table 8. Ethanol oxidation through photosensitized NAD+ regeneration in different photosystems ...

Fig. 3.4 The glycolytic pathway produces NADH which under regular conditions is oxidized to NAD+ while reducing acetaldehyde (ACA) to ethanol (EtOH), thereby in turn reducing NAD+ in order to keep hexose catabolism running. The actual cytosolic NADH concentration is determined by the respective conversion rates of the enzymes involved in the oxidation and regeneration of the compound. If these enzymes convert additional non-natural substrates (xenobiotics, i.e. drugs), the conversion rate changes. As a consequence, the cytosolic NADH concentration differs from the natural condition. Furthermore, if a xenobiotic acts as an enzyme inhibitor, e.g. for ADH, then NAD+ regeneration is substantially affected, which eventually results in altered cytosolic NADH concentration. Therefore the presence of a xenobiotic in the cell is conceivably a perturbation factor. Under the conditions where glycolytic oscillations...

Release of the chloride substituent in 2 produces ethyl acetoacetate (1) which is an inhibitor of alcohol dehydrogenase (ADH, EC 1.1.1.1) and thereby affects energy metabolism. Above all, NAD+ regeneration is impaired. As a consequence acetaldehyde becomes an overflow metabolite which is excreted into the medium or reacts with activated positions in the molecule (Fig. 3.5). 

Fig. 14 Kinetic resolution of or-fcrf-leucine catalyzed by L-leucine dehydrogenase (L-LeuDH) for the preparation of D-tert-leucine with simultaneous NAD+ regeneration using NADH oxidase (Nox) from Lactobacillus brevis...

Fig. 3.43 6-Methyl-hept-5-en-2-one reduction and simultaneous NAD+ regeneration using alcohol dehydrogenase from Rhodococcus rubber.

Mediators acting as one-electron transfer agents toward NADH must possess relatively positive potentials [107]. This is the case for the iron complexes mentioned earlier with potentials between 800 and 930 mV vs. NHE. The compound 2,2 -azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid)-diammonium salt, ABTS (shown here), which is mainly used as a redox-dye indicator for the activity determination of certain enzymes, can act as one-electron redox catalyst for the NAD" regeneration at somewhat lower potentials of 430 mV vs. Ag/AgCl [108]. 

The following data for the production of chiral y-lactones from me o-diols using the indirect electrochemical NAD" regeneration procedure can be given In a volume of 100 mL using 3.2 mg (1 x 10 mol) of PDMe, 70mg (1 x 10 mol)ofNAD, and 12.5mg(16U)of HLADH, after 20 h 99.5% of the meso-diol (0.5 to 1.0 g = 3.5 to 7.0 mmol) were converted to the enentiomerically pure lactone. Thus, the space-time yield can be calculated to be 6 to 12 g/L -day . The productivity is limited by the small amount of the enzyme. 

Figure 22. Glycerine dehydrogenase (GDH) catalyzed oxidation of mc.so-l,2-cyclohexandiol to give (S )-2-hydroxycyclohexanone with indirect electrochemical NAD" regeneration using PDMe as a redox catalyst.

NMPH reoxidation have been used as the measuring signal (Schubert et al., 1986b). In the latter system the amplification was significantly higher since the NAD+ regeneration proceeds in the whole volume of the enzyme membrane whereas the electrochemical oxidation of NADH takes place only at the electrode surface. 

Figure t. 2-5. intrasequerttfai NAD regeneration for HLADH-driven firs Stic racemate resolution esf ffrrydrojcysilanes. 

Alcohol dehydrogenases are generally applied for the interconversion of alcohols and aldehydes. Yet, these enzymes have also attracted interest due to their ability to oxidize aldehydes111. HLADH was shown to oxidize butanal[2l This reaction, however, shows no potential for synthetic application unless a very efficient NAD+ regeneration system is applied (Fig. 16.4-2). The catalytic activity of HLADH for the reduction of the aldehyde is more than 100 times higher than that for aldehyde oxidation (examined for benzaldehyde) 31. As a result, the initially formed NADH is... 

Finally, severe impairment of respiration impairs mitochondrial 6-oxidation (Watmough et al. 1990). Normally the NADH formed by 6-oxidation is then reoxidized by the mitochondrial respiratory chain regenerating the NAD" required for fatty acid 6-oxidation. During severe impairment of respiration, NAD" regeneration cannot sustain 6-oxidation (Watmough et al. 1990), which impairs 6-oxidation... 

More essential amino acids are produced by fermentation for the pharmaceutical markets - as ingredients in infusion solutions or as a precursor for pharmaceutical amino acid derivatives. The latter requires extremely ambitious bio-catalytic processes including co-factor regeneration. Evonik Industries developed a process to the pharma-building block L-tert. leucine based on formate-dehydrogenase and NAD+-regeneration (Figure 12.3). Today this process runs on a tons scale. 

The NAD regenerated by this reaction may then be used in the glycolysis pathway. 

Some species of the LAB group such as Leuconostoc mesenteroides subsp. cremoris, Leuconostoc mesenteroides subsp. dextranicum, and Lactococcus lactis subsp. lactis biovar diacetylactis, are known for their capability to produce diacetyl (2,3-butanedione) from citrate, and this metabolism appears especially relevant in the field of dairy products (Figure 13.4). Actually, selected strains belonging to the above species are currently added as starter cultures to those products, e.g., butter, in which diacetyl imparts the distinctive and peculiar aroma. Nevertheless, in particular conditions where there is a pyruvate surplus in the medium (e.g., in the presence of an alternative source of pyruvate than the fermented carbohydrate, such as citrate in milk or in the presence of an alternative electron acceptor available for NAD+ regeneration) (Axelsson, 2(X)9, pp. 1-72), even other LAB such as lactobacilli and pediococci can produce diacetyl by the scanted pyruvate (Figure 13.5). Thus, in addition to butter and dairy products, diacetyl can be present in other fermented foods and feeds, such as wine and ensilage (Jay, 1982). 

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