Coenzyme regeneration enzyme

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. 

Scheme 3.3 Coenzyme regeneration by a CRE (coenzyme regeneration enzyme)/BVMO fusion enzyme.

The second general approach is to use whole cells that contain the enzyme or enzymes used in the biocatalytic process. The use of whole cells has the added advantage that coenzyme-dependent enzymes can be used because it is possible to regenerate the relevant coenzyme, through metabolism of the whole cells. This, of course, requires that the whole cells are not only physically intact but also meta-bolically active. Since coenzymes are often involved in building new molecules, industrial biocatalysis typically uses whole-cell systems. 

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

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]. 

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...

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. 

Coenzymes - Many enzymes require nonprotein coenzymes for catalytic activity.8 These are cosubstrates, and must be constantly reconverted into their active form for catalysis to continue. This is not a problem for growing microorganisms since the normal metabolic processes ensure an adequate supply of coenzymes. However, with purified, or immobilized enzymes, maintaining a sufficient concentration of coenzyme can pose a major problem. Coenzymes are expensive and it is seldom economically feasible to add them in stoichiometric amounts. This is often undesirable for chemical reasons, e.g., the coenzyme may be unstable, or the eventual build-up of high concentrations of its inactive form may Induce displacement of an equilibrium reaction in the opposite direction to that desired.3 It is therefore necessary to use catalytic amounts of coenzymes and to ensure that the active forms are continuously regenerated. Some coenzymes present little or no problem in this regard since they are automatically reformed under the normal aqueous reaction conditions or in the presence of oxygen. These include biotin, pyrldoxal phosphate (PLP), thiamine pyrophosphate, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).1 ... 

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. 

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. 

Regeneration enzyme Cosubstrate/ coproduct Specific Activity [U mg 1] Stability Coenzyme E 0 [V] vs. NHEa i1 ... 

One elegant way of in situ product removal is to use the product of a first dehydrogenase reaction as substrate for a subsequent enzymatic reaction, thus recycling the oxidized nicotinamide coenzyme (Fig. 16.2-3). Various NAD(P)-de-pendent enzymes can be applied as regeneration enzymes in this cascade reaction. 

The application of enzyme systems, combining coenzyme regenerating reactions, has been studied by a group at the Vienna University of Agriculture. Typical examples are the simultaneous production of gluconic acid and xylitol [71] or sorbitol [72], respectively, in membrane reactors. 

P. Roehrig, C. M. Wolff, and J. P. Schwing, Repetitive Enzymatic Determination of Glucose with Regeneration and Recycling of Coenzyme and Enzymes. Anal. Chim. Acta, 153 (1983) 181. 

As more and more sophisticated systems were studied the demands placed on the immobilized enzyme preparation constantly increased. The low stability of intracellular enzymes as well as the problems encountered in coenzyme regeneration accelerated the development of immobilized microorganisms or organelles ( ). 

HLADH was used as redox catalyst in a coupled substrate-coenzyme regenerating cycle, and the enzymatic activity was studied as a function ofoil fraction of the microemulsion [118]. The oil fraction was varied while the surfactant (AOT) concentration was kept constant, leading to a change in the microstructure of the solution from an O/W to a W/O microemulsion via a bicontinuous structure, as determined by self-diffusion NMR. The enzyme exhibited good stability in the various types of structures. Variation of the initial reaction rate could be described by modifying the rate equation, valid in pure buffer, taking into account partitioning of the substrate between the water and hydrocarbon domains. 

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