Coenzyme regeneration methods

Efficient Coenzyme-Regeneration Methods Should be Available... 

Reduction of the substrate accompanies the oxidation of the coenzyme (Step 2). Before the next cycle of the reduction of the main substrate can occur, the coenzyme has to be reduced (Step 4). Many methods for the regeneration of the reduced form of coenzyme [NAD(P)H] have been developed, so that only a catalytic amount of the coenzyme is required for the reaction. The coenzyme regeneration methods can be classified into two types ... 

Alcohol Method for coenzyme regeneration Ketone concentration [mM] Yield [%] Enantiomeric excess [%] Ref. 

Generally, the nicotinamide coenzymes are not covalently bound to the enzyme. They are employed in enzyme assays and preparative applications by adding catalytical but optimized amounts, and they need to be recycled. For an economic process, an efficient regeneration method is a basic requirement. The necessary recycle number depends essentially on the value of the chiral product, generally the method should recycle the coenzyme 100-100,000 times ([42]). 

Limitations of the system are the low turnover numbers as well as the failure to regenerate NADPH. However, if ferredoxin reductase is used for coenzyme regeneration instead of lipoamide dehydrogenase, this method can also be applied to NADP+-dependent systems [97]. 

Breeze A.S. Simpson A.M. (1982) An improved method using acetyl-coenzyme A regeneration for the enzymic inactivation of aminoglycosides prior to sterility testing. JApplBacteriol, 53, 277-284. 

In fact, the a-ketoglutarate/glutamate dehydrogenase is a generally applicable method for the regeneration of NAD and NADP in laboratory scale productions. Both components involved are inexpensive and stable. Quite recently, a method for the oxidation of the reduced nicotinamide coenzymes based on bacterial NAD(P)H oxidase has been described [225], This enzyme oxidizes NADH as well as NADPH with low Km values. The product of this reaction is peroxide, which tends to deactivate enzymes, but it can be destroyed simultaneously by addition of catalase. The irreversible peroxide/catalase reaction favours the ADH catalyzed oxidation reaction, and complete conversions of this reaction type are described. 

A more promising approach for the synthesis of hydrophobic substances with ADHs is published by Kruse et al. [159, 238], They use a continuously operating reactor where the enzyme containing water phase is separated from the hydrophobic substrate-containing organic phase by a membrane. The hydrophobic product is extracted continuously via a hydrophobic membrane into an hexane phase, whereas the coenzyme is regenerated in a separate cycle, that consists of a hydrophilic buffer system. This method decouples advantageously the residence time of the cofactor from the residence time of the substrate. Several hydrophobic alcohols were prepared in this way with (S)-ADH from Rhodococcus erythropolis (Table 16). 

This review describes the use of different methods for the regeneration of nicotinamide coenzymes, particularly enzymatic and electrochemical strategies. 

In order to measure transhydrogenation spectrophotometrically without a regenerating system, coenzyme analogs, e.g., thio-NAD(P) (absorption maximum for the reduced form at 400 nm), have often been used (6, 20). However, coenzyme analogs are not natural substrates and therefore care should be taken in interpreting kinetic data obtained with these artificial substrates. On the other hand, the use of analogs appears to be the method of choice in cases where rapid reactions are measured or where low concentrations of products interfere (see 21). 

Recent Advancements in the Enzymatic Methods for the Recycling of NAD(P)(H) Coenzymes and Novel Regeneration Systems... 

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