Nicotinamide recycling

Each reaction was performed with a CYP biocatalyst concentration of 1 pM (1000 nmol L 1), in the presence of a corresponding CYP reaction mix containing reduced nicotinamide cofactor and a cofactor recycling system at 30 °C, with agitation to promote oxygen transfer to the reaction solution. 

The asymmetric reduction of prochiral functional groups is an extremely useful transformation in organic synthesis. There is an important difference between isolated enzyme-catalyzed reduction reactions and whole cell-catalyzed transformations in terms of the recycling of the essential nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] cofactor. For isolated enzyme-catalyzed reductions, a cofactor recycling system must be introduced to allow the addition of only a catalytic amount (5% mol) of NAD(P)H. For whole cell-catalyzed reductions, cofactor recycling is automatically achieved by the cell, and the addition of a cofactor to the reaction system is normally not required. 

It is possible to use isolated, partially purified enzymes (dehydrogenases) for the reduction of ketones to optically active secondary alcohols. However, a different set of complications arises. The new C H bond is formed by delivery of the hydrogen atom from an enzyme cofactor, nicotinamide adenine dinucleotide (phosphate) NAD(P) in its reduced form. The cofactor is too expensive to be used in a stoichiometric quantity and must be recycled in situ. Recycling methods are relatively simple, using a sacrificial alcohol, or a second enzyme (formate dehydrogenase is popular) but the real and apparent complexity of the ensuing process (Scheme 8)[331 provides too much of a disincentive to investigation by non-experts. 

Generally, the nicotinamide coenzymes are not covalently bound to the enzyme. They are employed in enzyme assays and preparative applications by adding catalytical but optimized amounts, and they need to be recycled. For an economic process, an efficient regeneration method is a basic requirement. The necessary recycle number depends essentially on the value of the chiral product, generally the method should recycle the coenzyme 100-100,000 times ([42]). 

Ketoreductases (KREDs) are dependent on nicotinamide cofactors NADH or NADPH. Due to the reaction mechanism, these rather costly cofactors are needed in stoichiometric amounts, disclosing an economic problem that has to be dealt with when using these enzymes. Many different possibilities for cofactor recycling have been established with three major approaches finding application in research and industry (Fig. 13). Further regeneration systems, such as electrochemical methods, are not discussed within this review [22-24, 37, 106-108],... 

For these reasons, recent efforts have been aimed at cheap, effident cofactor recycle systems for use with cell-free alcohol dehydrogenase preparations. Regeneration of the nicotinamide adenine dinudeotide cofactor required for alcohol synthesis can be catalyzed by a second enzyme or reduced by the same enzjme, provided the overall equilibrium is favorable. 

Formate dehydrogenase in conjunction with polyethyleneglycol-immobilized nicotinamide adenine dinudeotide has been used to good effect as a cofactor recycle system (39). The alcohol dehydrogenase from Thermoanaerobium hrockii catalyzed the reduction of ketones independently when driven by the cooxidation of isopropanol (40,41). 

The pentose phosphate pathway (PPP) is the major pathway for recycling nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and for the production of ribose-5-phosphate that is needed for the synthesis of nucleotides. The function of the PPP depends on the synthesis of nicotinamide-adenine dinucleotide phosphate (NADP) and thiamin pyrophosphate, a coenzyme... 

One elegant way of in situ product removal is to use the product of a first dehydrogenase reaction as substrate for a subsequent enzymatic reaction, thus recycling the oxidized nicotinamide coenzyme (Fig. 16.2-3). Various NAD(P)-de-pendent enzymes can be applied as regeneration enzymes in this cascade reaction. 

Among the chemical mediator systems especially o-quinones are capable of accepting the hydride equivalent from reduced nicotinamides. The oxidized mediators are regenerated by molecular oxygen. Since these mediators can also be recycled electrochemically, they are discussed in the following chapter. 

In contrast to the chemical alkaline hydrolysis of 3-cyanopyridine with 4% byproduct of nicotinic acid (96% yield) the biotransformation works with absolute selectivity and no acid or base is required. The biotransformation (a continuous process) is operated at low temperature and atmospheric pressure. In contrast to the old synthesis route of nicotinamide at Lonza, the new one is environmentally friendly and safe. There is only one organic solvent used throughout the whole process in four highly selective continuous and catalytic reactions. The process water, NH3 and H2 are recycled. 

The efficient utilization of the nicotinamide cofactors requires not only a scheme for their recycling but also a method for dealing with their limited lifetime in solution. A number of approaches to recycling have been considered, and some appear to be quite satisfactory. The only obvious approach to the economic problem posed by limited lifetime is to lower the initial cost of the cofactor, and we can only offer suggestions concerning this problem. ... 

In the energy metabolism of cells, an intermediate carrier of the electrons (and protons) is commonly required. Such an electron carrier is, for instance, NAD+ (nicotinamid adenin dinucleotide), which formally accepts two electrons and one proton and is thereby reduced to NADH. The NADH may give off the electrons again to specialized electron acceptors and the protons are released in the cell sap. Thereby, the NADH, which must be used repeatedly, is recycled. 

The xylose reductase (XR) catalyzes the first step of a fungal pathway that allows certain organisms to metabolize xylose, such as Candida boidinii [6], Candida guilliermondii [7], Candida tmpicalis [8], Candida parapsilosis [9], and Debaryomyces hansenii [10]. After the reduction of xylose to xylitol by XR in a manner that can utilize nicotinamide adenine dinucleotide (reduced form NADH) or nicotinamide adenine dinucleotide phosphate (reduced form NADPH), xylitol is re-oxidized to xylulose by xyUtol dehydrogenase, which is often specific for nicotinamide adenine dinucleotide (NAD) [11]. Xylulose is then phosphorylated. An efficient, pathway should recycle the cosubstrate such that there is no... 

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