NADP cofactor

The highly selective biocatalytic reactions afford a substantial reduction in waste. The overall isolated yield is greater than 90%, and the product is more than 98% chemically pure with an enantiomeric excess of >99.9%. All three evolved enzymes are highly active and are used at such low loadings that counter-current extraction can be used to minimize solvent volumes. Moreover, the butyl acetate solvent is recycled with an efficiency of 85%.The E factor (kgs waste per kg product) for the overall process is 5.8 if process water is excluded (2.3 for the reduction and 3.5 for the cyanation) [47]. If process water is included, the E factor for the whole process is 18 (6.6 for the reduction and 11.4 for the cyanation). The main contributors to the E factor are solvent losses which accounted for 51% of the waste, sodium gluconate (25%), NaCl and Na2SO4 (combined circa. 22%). The three enzymes and the NADP cofactor account for <1% of the waste. The main waste streams are aqueous and directly biodegradable. 

Maurer, S. C., Schulze, H., Schmid, R. D., and Urlacher, V. 2003. Immobilisation of P450BM-3 and an NADP(+) cofactor recycling system Towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv. Synth. Catalys.,345, 802-810. 

Figure 21. Photosensitized regeneration of NADP cofactors using 23 and 24 as photosensitizers.

The acetaldehyde dehydrogenase in S. cerevisiae has five isoforms, three located in the cytosol (Section 1.4.1) (Ald6p, Ald2p, and Ald3p) and the remaining two (Ald4p and AldSp) in the mitochondria (Section 1.4.3). These enzymes differ by their specific use of the NAD+ or NADP+ cofactor (Table 2.2). 

In oiological systems, the most frequent mechanism of oxidation is the remov of hydrogen, and conversely, the addition of hydrogen is the common method of reduc tion. Nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP) are two coenzymes that assist in oxidation and reduction. These cofactors can shuttle between biochemical reac tions so that one drives another, or their oxidation can be coupled to the formation of ATP. However, stepwise release or consumption of energy requires driving forces and losses at each step such that overall efficiency suffers. 

All dehydrogenations involve the transfer of two electrons (where 2H is equivalent to 2H plus 2e ). NAD" or NADP" are the cofactors in most substrate dehydrogenations these accept two electrons while a proton is released. 

For the majority of redox enzymes, nicotinamide adenine dinucleotide [NAD(H)j and its respective phosphate [NADP(H)] are required. These cofactors are prohibitively expensive if used in stoichiometric amounts. Since it is only the oxidation state of the cofactor that changes during the reaction, it may be regenerated in situ by using a second redox reaction to allow it to re-enter the reaction cycle. Usually in the heterotrophic organism-catalyzed reduction, formate, glucose, and simple alcohols such as ethanol and 2-propanol are used to transform the... 

Ketoreductases catalyze the reversible reduction of ketones and oxidation of alcohols using cofactor NADH/NADPH as the reductant or NAD + /NADP+ as oxidant. Alcohol oxidases catalyze the oxidation of alcohols with dioxygen as the oxidant. Both categories of enzymes belong to the oxidoreductase family. In this chapter, the recent advances in the synthetic application of these two categories of enzymes are described. 

Complex 9 (Scheme 43.3) can be reduced by different redox equivalents to the active rhodium(I) species 10 namely, by electrons, formate [37, 38], and hydrogen. This hydrido complex then transfers the hydride ion onto the nicotinamide. In electrochemical applications, TOFs in the range of 5 to 11 h-1 have been reported [31, 39]. It is noteworthy that this complex accepts NAD+ and NADP+ as substrates with the same efficiency and almost exclusively produces the 1,4-reduced cofactor (selectivity >99%). 

Until now, only a few versatile, selective and effective transition-metal complexes have been applied in nicotinamide cofactor reduction. The TOFs are well within the same order of magnitude for all systems studied, and are within the same range as reported for the hydrogenase enzyme thus, the catalytic efficiency is comparable. The most versatile complex Cp Rh(bpy) (9) stands out due to its acceptance of NAD+ and NADP+, acceptance of various redox equivalents (formate, hydrogen and electrons), and its high selectivity towards enzymatically active 1,4-NAD(P)H. 

This system fulfills the four above-mentioned conditions, as the active species is a rhodium hydride which acts as efficient hydride transfer agent towards NAD+ and also NADP+. The regioselectivity of the NAD(P)+ reduction by these rhodium-hydride complexes to form almost exclusively the enzymatically active, 1,4-isomer has been explained in the case of the [Rh(III)H(terpy)2]2+ system by a complex formation with the cofactor[65]. The reduction potentials of the complexes mentioned here are less negative than - 900 mV vs SCE. The hydride transfer directly to the carbonyl compounds acting as substrates for the enzymes is always much slower than the transfer to the oxidized cofactors. Therefore, by proper selection of the concentrations of the mediator, the cofactor, the substrate, and the enzyme it is usually no problem to transfer the hydride to the cofactor selectively when the substrate is also present [66]. This is especially the case when the work is performed in the electrochemical enzyme membrane reactor. 

Since many of the transformations undergone by metabolites involve changes in oxidation state, it is understandable that cofactors have been developed to act as electron acceptors/ donors. One of the most important is that based on NAD/NADP. NAD+ can accept what is essentially two electrons and a proton (a hydride ion) from a substrate such as ethanol in a reaction catalysed by alcohol dehydrogenase, to give the oxidized product, acetaldehyde and the reduced cofactor NADH plus a proton (Figure 5.2). Whereas redox reactions on metal centres usually involve only electron transfers, many oxidation/reduction reactions in intermediary metabolism, as in the case above, involve not only electron transfer but... 

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