NADH cofactor regeneration

An elegant four-enzyme cascade process was described by Nakajima et al. [28] for the deracemization of an a-amino acid (Scheme 6.13). It involved amine oxidase-catalyzed, (i )-selective oxidation of the amino acid to afford the ammonium salt of the a-keto acid and the unreacted (S)-enantiomer of the substrate. The keto acid then undergoes reductive amination, catalyzed by leucine dehydrogenase, to afford the (S)-amino acid. NADH cofactor regeneration is achieved with formate/FDH. The overall process affords the (S)-enantiomer in 95% yield and 99% e.e. from racemic starting material, formate and molecular oxygen, and the help of three enzymes in concert. A fourth enzyme, catalase, is added to decompose the hydrogen peroxide formed in the first step which otherwise would have a detrimental effect on the enzymes. 

Figure 5.9 NAD+/NADH cofactor regeneration a the coupled enzyme approach b the coupled substrate approach c, d examples [42,43] of the two approaches.

While this anode is not useful in the context of implantable fuel cells, it is of interest because methanol is an attractive anodic fuel due to its availability and ease of transport and storage. The oxidation of one equivalent of methanol requires the reduction of three equivalents of NAD+ to NADH. As the NADH cofactor itself is not a useful redox mediator, a benzylviologen/diaphorase redox cycle, with a redox potential of 0.55 V vs SCE at pH 7, was used to regenerate NAD+ for use by the dehydrogenases, as depicted in Fig. 12.10. 

Unlike the whole-cell system, enzymatic reductions require the addition of a hydride donating cofactor to regenerate the reduced form of the enzyme. Depending on the chosen ADH, the cofactor is usually NADH or NADPH, both of which are prohibitively expensive for use in stoichiometric quantities at scale. Given the criticality of cofactor cost, numerous methods of in situ cofactor regeneration, both chemical and biocatalytic, have been investigated. However, only biocatalytic regeneration has so far proven to be sufficiently selective to provide the cofactor total turnover numbers of at least 10 required in production. 

In contrast, amino acid dehydrogenases comprise a well-known class of enzymes with industrial apphcations. An illustrative example is the Evonik (formerly Degussa) process for the synthesis of (S)-tert-leucine by reductive amination of trimethyl pyruvic acid (Scheme 6.12) [27]. The NADH cofactor is regenerated by coupling the reductive amination with FDH-catalyzed reduction of formate, which is added as the ammonium salt. 

Biocatalysts based on hydrolases (E.C. class 3, Table 5.2) ate mostly used as (purified) enzymes since they are cofactor independent, since these preparations are commercially available and because a number of hydrolases can be applied in organic solvents. Oxidoreductases (E.C. class 1) however, are relatively complex enzymes, which require cofactors and frequently consist of more than one protein component. Thus, despite the fact that efficient cofactor regeneration systems for NADH based on formate dehydrogenase (FDH) have been developed (Bradshaw et al, 1992 Chenault Whitesides, 1987 Wandrey Bossow, 1986, chapter 10) and that also an NADPH dependent FDH has been isolated (Klyushnichenko, Tishkov Kula, 1997), these enzymes are still mostly used as whole-cell biocatalysts. 

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. 

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... 

Both competing reductions consume the cofactor nicotinamide adenine dinucleotide (NADH) and thereby interfere with the redox balance of the cell and feedback on glycolysis where NADH is regenerated on the one hand, while on the other hand NAD+ is required to keep the glycolytic pathway running. The nonlinear dynamical model combines the network of glycolysis and the additional pathways of the xenobiotics to predict the asymmetric yield (enantiomeric excess, ee) of L-versus D-carbinol for different environmental conditions (Fig. 3.4). Here, the enantiomeric excess of fluxes vy and i>d is defined as... 

Scheme 4.101). The cofactor NADH was regenerated by the simultaneous oxidation of isopropanol to acetone. 

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... 

Heterofermentative LAB have the capability to utilize high concentrations of fructose such that the mannitol concentration in the fermentation broth could reach more than 180g/L, which is enough to be separated from the cell-free fermentation broth by cooling crystallization. Lactic and acetic acids can be recovered by electrodialysis (Soetaert et al., 1995). The enzyme mannitol dehydrogenase responsible for catalyzing the conversion of fructose to mannitol requires NADPH (NADH) as cofactor. Thus, it is possible to develop a one-pot enzymatic process for production of mannitol from fructose if a cost-effective cofactor regeneration system can be developed (Saha, 2004). The heterofermentative LAB cells can be immobilized in a suitable support, and... 

The enzyme hydrogenase (hydrogen dehydrogenase EC 1.12.1.2) is able to reduce electron acceptors by molecular hydrogen. When it is used in cofactor regenerating systems, consumed NADH can be regenerated directly by molecular hydrogen. 

Fig. 13 Production of (S)-1 -phenylethanol from rac-1 -phenylethanol catalyzed hy (/J)-specific ADH with coupled cofactor regeneration using NADH oxidase from Lactobacillus brevis...

In another approach, Yoneyama used electrogenerated and regenerated 1-phenyl-ethanol as reducing equivalent for the alcohol dehydrogenase-catalyzed regeneration of NADH. In this system, the production enzyme and the cofactor regenerating enzyme are identical. A production of 2 pmol was reached at turnover frequency of 1.7 TN/h or less (Fig. 9) [46]. 

The reaction is catalyzed by D-hydroxyisocaproate dehydrogenase (D-HicDH). The essential cofactor PEG-NADH is regenerated from PEG-NAD+ by a second enzyme, formate dehydrogenase (FDH). By coupling to water-soluble polyethyleneglycol with a molar mass of 20 000 g mol-1, the cofactor can be retained, together with the enzymes, by an ultrafiltration membrane, and the whole process may be performed continuously in an enzyme membrane reactor. 

Here we divide the discussion of approaches to cofactor regeneration into three sections one each for ATP, oxidized nicotinamide cofactors (NAF " and NADP ), and reduced nicotinamide cofactors (NADH and NADPH). Most of the other cofactors which appear in biochemistry are either easily regenerated or of little importance, and we shall not discuss their regeneration here. 

In K. pneumoniae, 3-HP and PDO are derived from the same intermediate, 3-HPA. The fate of 3-HPA depends on the activity of relevant enzymes (oxidoreductases and 3-HPA-specific ALDH) and the availability of redox cofactors (NAD" " and NADH). If a single product, either 3-HP or PDO, should be produced at high yield, one of the other types of enzymes should be disrupted and one kind of cofactor should be regenerated efficiently. When compared to gene disruptions, cofactor regeneration is much more challenging. In K. pneumoniae, cofactor regeneration is performed by oxidative... 

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