Nicotinamide recycling system

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. 

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 main problem of the co-TA-catalyzed transamination is the generally unfavorable equilibrium that lies on the substrate side, especially when alanine is used as amino donor. Several methods have been successfully established to overcome this issue, for instance, coproduct removal [e.g., with lactate dehydrogenase (LDH)] or amino donor recycling [e.g., with alanine dehydrogenase (AlaDH)] [142-145]. The nicotinamide cofactor consumed in this reductive step is regenerated by an additional recycling system. These multienzymatic systems based on co-TAs were applied to the synthesis of biologically active compounds. 

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],... 

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. 

Scheme 4.10 Synthesis of interesting building blocks using an ADH plus AlDH system to recycle the nicotinamide cofactor.

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