Enzymes regenerating systems

Assay of AB-specific transhydrogenase is principally identical to that of BB-specific transhydrogenase (see Section II,C) i.e., with the natural nicotinamide nucleotides, an enzymic regenerating system is used to keep the concentration of one of the substrates constant (30, SB, 66-68, 66)... 

Figure 2. Principle of an enzyme-coupled cofactor or enzyme regeneration system for an enzymatic reduction process.

A lot of analytical techniques have been proposed in recent decades and most of them are based on enzymes, called dehydrogenases, which are not sensitive to oxygen and need cofactors such as NAD". The key problems which seriously hamper a wide commercialization of biosensors and enzymatic kits based on NAD-dependent enzymes are necessity to add exogenous cofactor (NAD" ) into the samples to be analyzed to incorporate into the biologically active membrane of sensors covalently bounded NAD" to supply the analytical technique by NAD -regeneration systems. 

When the reaction product is soluble in water, enzyme regeneration is difficult to achieve, since the enzyme is often lost during isolation of the product. One way to overcome this problem is application of immobilised enzyme systems. The enzyme is either covalently or ionically attached to an insoluble carrier material or is entrapped in a gel. Depending on the size of the particles used, a simple filtration and washing procedure can be used to separate the immobilised enzyme from the dissolved product A well-known example of this technique is the industrial production of 6-APA. 

Rupprath, C., Kopp, M., Hirtz, D. et al. (2007) An enzyme module system for in situ regeneration of deoxythymidine 5 -diphosphate (dTDP)-activated deoxy sugars. Advanced Synthesis Catalysis, 349, 1489-1496. 

Formally, in its oxidized state the cofactor NAD+ is charged negatively due to the two phosphate groups the positive charge denotes quaternization of the nitrogen. It is noteworthy that from the reduced form only the 1,4-NAD(P)H instead of the 1,6-NAD(P)H is enzyme-active, which imposes some restrictions on the regeneration systems in terms of the selectivity. 

Enzymatic cofactor regeneration can be subdivided into two categories the enzyme-coupled approach, where two different enzymes are used (one for the production reaction, and one for the regeneration reaction) and the substrate-coupled approach, where one and the same enzyme is used for both production and regeneration (E = E2). The most convenient and commonly used enzymatic regeneration systems are summarized in Table 43.1. 

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. 

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],... 

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