Regeneration coenzyme

A number of photochemically or photoelectrochemically activated transition-metal complexes have also been used, both for oxidation and reduction of the nicotinamide cofactors. Among these complexes is the aforementioned Cp Rh(bpy)-complex 9 [52, 53]. For details of these systems or other regeneration procedures using special dyes, the reader is referred to other reviews on coenzyme regeneration [17, 21-23]. 

K. Nakamura, M.Aizawa and O.Miyawaki, Electroenzymology, Coenzyme Regeneration, Springer Verlag, Berlin (1988). 

Leonida, M.D., Redox enzymes used in chiral syntheses coupled to coenzyme regeneration. Curr. Med Chem., 2001, 8, 345-369. 

Nakamura, K. Aizawa, M. Miyawaki, O. Electro-enzymology, Coenzyme Regeneration, SpringerNer a%. Berlin, 1988. 

B. Nidetzky, K. Schmidt, W. Neuhau-SER, et al, Application of charged ultrafil-tration membranes in continuous, enzyme-catalysed processes with coenzyme regeneration, in Separations for Biotechnology 3 (ed. D. L. Pyle) Royal Society of Chemistry, 1994, 351. 

An example of coenzyme regeneration with isolated enzymes is L-alanine production from pyruvate with an NADH-dependent alanine dehydrogenase (AlaDH) ... 

Usually a large number of other reactions will occur simultaneously, some of them being beneficial for the coenzyme regeneration, whereas others lead to undesired byproducts. Also, the substrate and prodnct of the main reaction may get involved in undesirable side-reactions. Therefore, whole cell reactions may be cheaper and simpler to carry out than reactions using isolated enzymes, bnt they are less easily controlled, less reproducible and yield more waste. The most widely stndied class of reactions nsing whole cells are redactions catalyzed by baker s yeast, which is cheap and widely available (Sybesma et al 1998). 

A set of Saccharomyces cerevisiae reductases was screened in collaboration with J. D. Stewart s group (University of Florida). Itwas demonstrated that diketo ester la is accepted as substrate by at least three different NADP(H)-dependent reductases of this microorganism. Application of a cell-free system in preparative batches using enzyme-coupled coenzyme regeneration afforded (R)-2a with more than 99% enantiomeric excess [13]. 

Efficient Coenzyme-Regeneration Methods Should be Available... 

Table 8. Preparation of chiral alcohols by enzyme-catalyzed reduction of the corresponding ketones with ADH from Lactobacillus kefir. The production of phenylethanol with formate and formate dehydrogenase (FDH) for coenzyme regeneration was carried out continuously in an enzyme-membrane-reactor...

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

In general, syntheses with isolated enzymes can be performed with higher selectivity and space-time yield than with whole cells, but they require in any case the coupling of coenzyme regenerating reactions. 

Keywords Chiral compounds, Dehydrogenases, Enzymatic reduction, Nicotinamide coenzymes, Regeneration of coenzymes... 

Fig. 12 Oxidation of cyclohexanol catalyzed by HLADH with acetaldehyde as a coenzyme regenerating substrate...

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]. 

Fig. 36 Conversion of 2-keto-6-hydroxyhexanoic acid to L-6-hydroxynorleucine. Reductive ami-nation is carried out by glutamate dehydrogenase (GluDH), coenzyme regeneration by glucose dehydrogenase (GDH)...

In this particular case, we decided to take advantage of the favorable thermodynamic equilibrium constant that drives the oxidation of phosphite to phosphate mediated by a recently described phosphite dehydrogenase (PTDH) [117] to a nearly irreversible process [118]. The exquisite selectivity of PTDH for phosphite also precludes any side reaction that can occur in case, for example, an ADH is used. These characteristics render PTDH as an ideal candidate for use as a coenzyme regenerating enzyme (GRE) in combination with BVMOs or other NAD(P)H-dependent enzymes. 

Structure of the reaction system. The term structure should include the sum of the chemical reactions occurring within the reaction system, e.g. parallel reactions, consecutive reactions (see Eq. (4)), coupled reactions such as in the case of coenzyme regeneration (see Eq. (49)), non-catalyzed reactions occurring alongside the enzymatic reaction. 

Dealing with complex systems (two or more coupled enzymatic reactions or reactions with coenzyme regeneration) a complete kinetic investigation and computer simulation of the reaction system is very helpful to achieve the desired selectivity and yield of reaction (e. g. by choosing a sensible substrate and coenzyme concentration, enzyme ratio and reaction time). A case study is available[42, 431 exemplifying the production of L-tert-leucine by reductive animation and simultaneous coenzyme regeneration. 

Considerations about process optimization of coupled systems with coenzyme regeneration are discussed in the literature(29, 42,43, 13S. One aspect may be illustrated here - the question of enzyme ratio within the coupled enzyme system. 

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 ... 

Not only the enzymes but also the cellular components such as coenzymes and carbohydrates are conserved in the cell, which makes the whole cell processes favorable. For example, the addition of an expensive coenzyme and an auxiliary enzyme for coenzyme regeneration is not necessary, which makes the system simple and economical when comparing with the equivalent isolated enzyme process. 

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