Cofactor regeneration systems, for

Fig. 2, Principal of an enzyme-coupled cofactor regeneration system for an enzymatic reduction...

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. 

Figure 19.5. Cofactor regeneration systems for NAD(P)H-dependent enzyme reactions. The enzyme-coupled one involving GDH (a), that involving FDH (b), and the substrate-coupled one (c). AR1, aldehyde reductase from S. salmonicolor Leu DH, leucine dehydrogenase ADH, sec-alcohol dehydrogenase.

Figure 4 (a) Regeneration systems for UDP-sugar cofactors. E, glycosyltransferase Ey pyruvate... 

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... 

Besides the oxygenating enzyme, NADPH and oxygen, the procedure utilizes a regeneration system for reduced nicotinamide cofactor that is based on glucose-6-phosphate and glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides394. 

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. 

Figure 3. Principle of an indirect electrochemical cofactor or enzyme regeneration system for an enzymatic reduction process.

Table 2 Comparison of Different Cofactor Regeneration Systems, FMN/O2, Ru(PD)3/02, and PDME" /Anode, for the Enzymatic Oxidation of / e o-3,4-Dihydroxymethlcyclohexene Catalyzed by HLADH [125]...

This class of reagents holds the most promise for rapid development in the near future as most reactions are asymmetric. The problems being overcome are the tight substrate specificity of many enzymes and the need for cofactor regeneration. Systems are now being developed for asymmetric synthesis rather than resolution approaches. Some of these reactions are discussed in Chapter 13. 

Complex Multicomponent Cofactor Regenerating Systems More complex multi-component cofactor regeneration systems have been described for nonfused P450 enzymes, which are accompanied by electron transfer between independent redox partners. The best characterized system in this respect is P450 from Pseudomonas... 

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