Cofactor regeneration systems

Biocatalytic ledox reactions offer great synthetic utility to organic chemists. The majority of oxidase-catalyzed preparative bioconversions are still performed using a whole-ceU technique, despite the fact that the presence of more than one oxidoreductase in cells often leads to product degradation and lower selectivity. Fortunately, several efficient cofactor regeneration systems have been developed (160), making some cell-free enzymatic bioconversions economically feasible (161,162). 

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

Glucose-6-phosphate dehydrogenase, cofactor regenerating system, 3 673... 

FADH2 halogenases such as tryptophan 7-halogenase have been shown to catalyze regioselective halogenation of a wide range of indole derivatives and aromatic heterocycles, where a cofactor regeneration system has recently been developed to use only a catalytic amount of cofactors (Scheme 7.13) [52, 53]. 

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. 

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which N-acetylneuraminic acid (NeuAc) is generated in situ from IV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

Fig. 22. Schematic presentation of the enzymatic synthesis of UDP-GalNH2 (33) including cofactor regeneration systems. A nucleoside monophosphate kinase (EC 2.7.7.4), B sucrose synthase (EC 2.4.1.13), C gal-l-P uridyltransferase (EC 2.7.7.12), D phosphoglucomutase (EC 2.7.5.1), E glucose-6-P dehydrogenase (EC 1.1.1.49), F lactate dehydrogenase (EC 1.1.1.27), G pyruvate kinase (EC 2.7.1.40) [319]...

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.

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. 

CRS = cofactor regeneration system Figure 20.5 Overview of monooxygenase reactions in synthetic chemistry. 

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. 

Ito and Paulson designed a cofactor regeneration system that overcomes several limitations of rran -sialidase-catalyzed sialylations (Scheme 45) [58]. The trans-sialidase is used in conjimction with a-2,3-sialyltransferase to effectively broaden the... 

The use of isolated enzymes to form or cleave P-O bonds is an important application of biocatalysts. Restriction endonucleases, (deoxy)ribonucleases, DNA/ RNA-ligases, DNA-RNA-polymerases, reverse transcriptases etc. are central to modern molecular biology(1). Enzyme catalyzed phosphoryl transfer reactions have also found important applications in synthetic organic chemistry. In particular, the development of convenient cofactor regeneration systems has made possible the practical scale synthesis of carbohydrates, nucleoside phosphates, nucleoside phosphate sugars and other natural products and their analogs. This chapter gives an overview of this field of research. 

Several cofactor regeneration systems were based on FDH. The substrate formate is an inexpensive, stable, and innocuous compoimd, while CO2, which is produced by FDH, can be easily removed from the reaction by evaporation. A general drawback of FDH is, however, its low specific activity [262]. More stable FDH variants have been engineered and successfully applied... 

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