NADH regeneration

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

Formate dehydrogenase can be said to catalyze a kind of decarboxylation reaction and is the most widely used in NADH regeneration. However, as the reaction does not include C—C bond fission, the studies on this enzyme are not described in this chapter. 

Sambongi Y, Nitta H, Ichihashi K, Futai M, Ueda I (2002) A novel water-soluble Hantzsch 1,4-dihydropyridine compound that functions in biological processes through NADH regeneration. J Org Chem 67 3499-3501... 

In an early report to a process using three oxidoreductases, namely hydrogenase (ECl.12.2.1), lipoamide dehydrogenase (EC 1.6.4.3) and 20(3-hydroxysteroid dehydrogenase (ECl.1.1.53), a reverse micelle system was used to facilitate stereo- and site-specific reduction of apolar ketosteroids, assisted by the in situ NADH-regenerating enzyme system [61]. 

Several biotransformation processes were developed. As an example Allelix used screening to isolate a Corymbacterium capable of converting df-acetamidocirmamic acid into L-phenylalanine. Lactate was added to the media to stimulate NADH regeneration. The key enzymes involved were cloned to increase copy numbers, the cells were used in iimnobilized form and phenylalanine recovery was facilitated by precipitation techniques. This approach was also explored by the Tanabe Co. in Japan. By contrast Cenex used the phenylalanine arrrmonia lyase activity of Rhodococcus rubra to convert irans-cinnamic acid. 

Since FDH from Candida boidinii now can be produced at pilot scale, this reaction can be generally used for NADH-regeneration. Recently, the same concept has been used for NADPH regeneration. This became possible because a NADPH-dependent FDH has been obtained by multipoint site-directed mutagenesis of the gene coding the enzyme from the bacterium sp. 101. (Seelbacheia/., 1996). 

M.-R. Kula and C. Wandrey, Continuous enzymic transformation in an enzyme-membrane-reactor with simultaneous NADH regeneration, Meth. Enzymol. 1987, 136, 9-21. 

Enzymatic synthesis of E-tm-leucine is another example of the use of isolated enzymes (Bommarius et al, 1995). An NADH-dependent leucine dehydrogenase was used as a catalyst for the reductive amination of the corresponding keto acid together with formate dehydrogenase (FDH) and formate as a cofactor regenerator (Fig. 19.5b Shaked and Whitesides, 1980 Wichmann et al, 1981). Furthermore, a unique membrane reactor system involving FDH and PEG-modihed-NAD for continuous NADH regeneration... 

Shaked, S., and Whitesides, G. M. 1980. Enzyme-catalyzed organic synthesis NADH regeneration by using formate dehydrogenase. J. Am. Chem. Soc., 102, 7104-7105. 

In both steps, commercially available GDH was used for NADH regeneration [47]. 

Fig. 31 Preparation of optically active compounds employing HLADH and NADH, which are codeposited onto glass beads in a monophasic organic solvent, (a) Reduction reaction to produce chiral alcohols in the presence of ethanol for NADH regeneration, (b) Oxidation reaction to produce enantiomerically pure alcohol or a ketone out of the racemic mixture coupled with the reduction of isobutyraldehyde to regenerate NAD+...

To catalyze the reduction of cyclohexanone to cyclohexanol in an organic-aqueous two-phase system, soluble hydrogenase from A. eutrophus as an NADH regenerating catalyst was coupled with HLADH (Fig. 32) [37]. 

Several organic solvents were investigated with regard to stability and activity of HLADH as well as their influence on the hydrogenase-driven reaction. Hydrophobic solvents such as heptane or toluene proved to be the most suitable solvents for the coupled enzyme-system. Furthermore, it became apparent that nonimmobilized cells, permeabilized with cetyl-trimethylammonium bromide, showed the best results for NADH regeneration. After optimization the conversion in heptane with 10% water yields 99% cyclohexanol by reduction of cyclohexanone. 

Ru(bpy)3" ]. This reduced species is oxidized by Rh(bpysa).r, resulting in the formation of the reduced hydridorhodium complex, which provides further reduction of NAD" ". The NADH regenerated in this photochemical system was coupled with the enzymatic reduction of acetaldehyde to ethanol in the presence of alcohol dehydrogenase. 

In a different approach, instead of methyl viologen, the organic disulfide dithiothrei-tol was cathodically reduced to give the bis-thiol, which consecutively reduces the disulfide bridge within the lipoamide of the lipoamide dehydrogenase. This is followed by the NADH regeneration by the reduced LipDH in combination with the production of enzyme L-lactate dehydrogenase for the production of L-lactate from pyruvate. Both enzymes were immobihzed within a poly (acrylamide) gel [50]. 

It has also been pointed out that electrogenerated FMNH2 in combination with diaphorase can also act as mediator for the NADH regeneration in an uphill reaction [51]. 

In a different approach, instead of using a production enzyme together with an NADH-regenerating enzyme, baker s yeast was used to take over both objectives. Thus 3-keto esters were electrochemically reduced to give the optically active 3-hydroxy esters in the presence of baker s yeast and NAD" " using a viologen as redox catalyst to shuttle the electrons from the cathode to the yeast cells which then catalyze the NADH formation and the enzymatic reduction. In such an approach, usually the permeation in and out of the yeast cells is a limiting factor [54]. 

Figure 11. Hydrogenase-catalyzed NADH regeneration coupled to the reductive amination of a-ketoglutarate to L-glutmate catalyzed by L-glutamate dehydrogenase.

Figure 12. o-Lactate generation from pyruvate catalyzed by o-lactate dehydrogenase (d-LDH) with in situ indirect electrochemical NADH regeneration using a [Cp Rh(bpy)X] complex as a selective mediator [62]. 

Figure 13. Continuous formation of (S )-4-phenyl-2-butanol from 4-phenyl-2-butanone using the electrochemical enzyme membrane reactor under indirect electrochemical NADH regeneration with a high-molecular-weight rhodium catalyst [26,29,30,65].

Figure 1-9. NADH regeneration using formate dehydrogenase (FDH) in a coupled reaction with leucine dehydrogenase (LeuDH).

Figure 15.3-5. Enzymatic routes to L-phenylalanine via phenylpyruvate191. (i) Reductive animation of phenylpyruvate by PheDH with simultaneous NADH regeneration using FDH.

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