Regeneration of NADH

Figure 8.9 Regeneration of NADH using diaphoras and electric power. Dl diaphorase [7a].

Figure 8.10 Regeneration of NADH with rhodium complex using electric power [7bj.

Electrocatalytic regeneration of NADH has also been performed at polymer-modified electrodes with pendent [(r/s-Cs M cs) R h( bpy )C1]1 55,56 and [Rh(terpy)2]3+ 57 complexes, and coupled to enzymatic reduction with alcohol dehydrogenase.55,57... 

Fig. 17. Electroenzymatic reduction of 4-phenyI-2-butanone catalyzed by HLADH with in-situ indirect electrochemical regeneration of NADH using a Cp (2,2 -bipyridyl)aquo rhodium(III) complex as mediator...

When the resulting mixture of benzoylformic acid and (i )-mandelic acid was treated with a cell free extract of Streptomyces faecalis IFO 12964 in the presence of NADH,the keto acid can be effectively reduced to (i )-mandelic acid (Fig. 1). Fortunately the presence of A. bronchisepticus and its metabolite had no influence on the reduction of the keto acid. The regeneration of NADH was nicely achieved by coupling the reaction with reduction by formic acid with the aid of formate dehydrogenase. As a whole, the total conversion of racemic mandelic acid to the i -enantiomer proceeded with very high chemical and optical yields. The method is very simple and can be performed in a one-pot procedure [6]. 

The advantage of this reaction is that lactate is mnch cheaper than pyruvate, so the reaction is not only useful for the regeneration of NADH but also for the production of pyruvate. The disadvantage is that lactate dehydrogenases are enantiospecific, so a D-lactate as well as an L-lactate dehydrogenase are reqnired for a full conversion of racemic lactate (Figure 10.7). 

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. 

There are maty other examples of cofactor regeneration reactions and/or of reactions which may be performed in an enzyme membrane reactor. An important example is the regeneration of NADH by formate dehydrogenase (FDH), starting with formate (Wichmaim et al, 1981). The advantage of this reaction is that it is irreversible because carbon dioxide is hberated, while formate is a relatively cheap electron donor. 

Scheme 7. Indirect electrochemical regeneration of NADH from NAD using [Rhibpy-S-SS),] as redox catalysts...

The negative effects of acetyl-CoA on pyruvate dehydrogenase activity are supplemented by ATP and NADH. These effects are in the correct direction to cause the rate of acetyl-CoA synthesis to vary with the need for electrons and for regeneration of NADH and ATP. 

Several chemical routes have been developed for the regeneration of NADH and NADPH (25, 26). We have developed (27) a photochemical system for regeneration of NADPH (Figure 9). 4,4 -Bi-... 

R)-2-Hydroxy-4-phenylbutyric acid was produced continuously in an enzyme membrane reactor by enzymatic reductive animation of the a-keto acid with d-lactate dehydrogenase coupled with formate dehydrogenase (FDH) for regeneration of NADH. Reactor performance data matched a kinetic reactor model (Schmidt, 1992). 

For the regeneration of NADH, formate and formate dehydrogenase are most widely used. The enzyme from Candida boidinii is commercially available, formate is inexpensive, and the equilibrium favours a nearly irreversible... 

HLADH was applied for the reduction of a broad variety of substrates. The regeneration of NADH can be achieved by the ethanol coupled-substrate method [239]. Cyclic ketones are good substrates for this enzyme [137, 240, 241]. Heterocyclic compounds containing oxygen or sulfur are accepted, too [242 246], but nitrogen-containing substrates are inhibitors of HLADH, because they apparently complex with zinc in the active site of the enzyme [247],... 

SB Sobolov, MD Leonida, A Bartoszko-Malik, KI Voivodov, F McKinney, AJ Fry. Cross-linked LDH crystals for lactate synthesis coupled to electroenzymatic regeneration of NADH. J Org Chem 61 2125-2128, 1996. 

Fig. 15a, b. Biocatalyzed tranformations coupled to the regeneration of cofactors a) reduction of C02 to formate through regeneration of acetyl-CoA b) reduction of pyruvic acid to lactic acid by regeneration of NADH cofactor... 

The analysis also revealed 20 other elementary modes of degradation of 2 that do consume NADH and therewith interfere with glycolysis. In order to facilitate these additional modes for a longer period of time, the cell has to ensure the necessary regeneration of NADH which under anaerobic conditions may proceed via the secretion of acetaldehyde or succinate. The analysis has shown that production of cytosolic intermediates along this large set of flux modes appears likely on the basis of the stoichiometric data. 

Scheme 4.102 Indirect chemical regeneration of NADH using FAD/FADH2 as the mediator and subsequent biocatalytic conversion [425]. Reprinted with permission from [425]. Copyright 2005 American Chemical Society.

Shi, Q., D. Yang, Z.Y. Jiang and J. Li (2006). Visible-light photocatalytic regeneration of NADH using p-doped Ti02 nanoparticles. Journal of Molecular Catalysis B-Enzymatic, 43(1-4), 44 18. 

In particular, FDH from Candida boidinii is often used for the regeneration of NADH. However, the low specific activity of this enzyme (4-6 U mg-1) is a considerable disadvantage. The KM values for NAD+ and formate are 0.09 and 13 mM, respectively. The enzyme has a broad pH optimum of 7.5-8.5 while 55°C is the optimal temperature [3]. 

Fig. 3 Preparation of D-lactate from pyruvate catalyzed by d-LDH (D-lactate dehydrogenase) using formate/FDH for the regeneration of NADH...

Fig. 8 Synthesis of amino acids by a multienzyme system consisting of leucine dehydrogenase (LeuDH) catalyzing the reductive amination of the corresponding keto acid, L-lactate dehydrogenase (l-LDH), and lactate for the regeneration of NADH and urease for the in situ generation of ammonia. The coenzyme NAD+ was covalently bond to dextran, enzymes and dextran-coupled NAD+ were...

Fig. 21 l-LDH catalyzed reduction of pyruvate coupled with regeneration of NADH based on electrochemical reduction of disulfides. LipDH Lipoamide Dehydrogenase DTT Dithiothreitol Lip Lipoamide (R = (CH2)4CONH2)... 

In another system, the [Cp Rhm(bpy)(H20)]2+ complex was used for the regeneration of NADH in combination with an isolated flavine-dependent monooxygenase (2-hydroxybiphenyl-3-monooxygenase) from P. azelaica [120]. This enzyme catalyzes the specific ort/m-hydroxylation of several a-substituted phenol derivatives with simultaneous oxidation of NADH (Fig. 25). 

Fig. 26 Regeneration of NADH by the [Cp Rh(bpy)(H20)]2+ complex. The corresponding hydridorhodium complex can be generated either electrochemically by cathodic reduction (method a) or chemically with formate (method b). (Cp = C5Me5 bpy = 2,2-bipyridine)...

Fig. 27 Conversion of a-ketoglutarate to L-glutamate catalyzed by GluDH with coupled electrochemical regeneration of NADH using hydrogenase from Alcaligenes eutrophus...

Fig. 32 Reduction of cyclohexanone catalyzed by HLADH with simultaneous hydrogenase-driven regeneration of NADH in an organic-aqueous two-phase system...

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