Regeneration enzyme

Coexpression of Genes for Carbonyl Reductase and Cofactor-Regenerating Enzymes... 

Figure 8.15 Coexpression of genes for carbonyl reductase and cofactor-regenerating enzymes [llc,dj.

Let us consider the basic enzyme catalysis mechanism described by the Michaelis-Menten equation (Eq. 2). It includes three elementary steps, namely, the reversible formation and breakdown of the ES complex (which does not mean that it is at equilibrium) and the decomposition of the ES complex into the product and the regenerated enzyme ... 

Anaerobic bio-reduction of azo dye is a nonspecific and presumably extracellular process and comprises of three different mechanisms by researchers (Fig. 1), including the direct enzymatic reduction, indirect/mediated reduction, and chemical reduction. A direct enzymatic reaction or a mediated/indirect reaction is catalyzed by biologically regenerated enzyme cofactors or other electron carriers. Moreover, azo dye chemical reduction can result from purely chemical reactions with biogenic bulk reductants like sulfide. These azo dye reduction mechanisms have been shown to be greatly accelerated by the addition of many redox-mediating compounds, such as anthraquinone-sulfonate (AQS) and anthraquinone-disulfonate (AQDS) [13-15],... 

Scheme 43.2 Principal strategies of cofactor regeneration (En production enzyme E2 regeneration enzyme).

Indirect Electrochemical NAD(P)H Regeneration Without a Regeneration Enzyme... 

To be able to regenerate NADP(H) by an indirect electrochemical procedure without the application of a second regeneration enzyme system, the redox catalyst must fulfill four conditions ... 

In an early report to a process using three oxidoreductases, namely hydrogenase (ECl.12.2.1), lipoamide dehydrogenase (EC 1.6.4.3) and 20(3-hydroxysteroid dehydrogenase (ECl.1.1.53), a reverse micelle system was used to facilitate stereo- and site-specific reduction of apolar ketosteroids, assisted by the in situ NADH-regenerating enzyme system [61]. 

Pyridoxal phosphate (aldimine form, on regenerated enzyme)... 

In models for carboxypeptidase A we showed the intracomplex catalyzed hydrolysis of an ester by a metal ion and a carboxylate ion [106], which are the catalytic groups of carboxypeptidase A. Some mechanistic proposals for the action of carboxypeptidase involve an anhydride intermediate that then hydrolyzes to the product and the regenerated enzyme. Although we later found convincing evidence that the enzyme does not use the anhydride mechanism in cleaving peptides [96-99], it may well use such a mechanism with esters. In a mimic of part of this mechanism we showed [107], but see also Ref. 108, that we could achieve very rapid hydrolysis of an anhydride by bound Zn2+, which is the metal ion in the enzyme. In another model, a carboxylate ion and a phenolic hydroxyl group, which are in the enzyme active site, could cooperatively catalyze the cleavage of an amide by the anhydride mechanism [109]. 

An increasing number of examples in technical scale applications are known where nicotinamide dependent enzymes were used together with cofactor regenerating enzymes. 

A further important aspect is the feasibility of whole cell biotransformations. Whole cell biotransformations show a lot of advantages as compared to isolated enzymes, such as the improved stability of enzymes. If both, producing and regenerating enzymes, are available in one single strain, no addition of expensive cofactor is necessary because the intracellular cofactor pool can be utilized. Whole cell biotransformations are therefore very promising for technical applications, and making these conversions an intensively studied subject in the last years. The use of recombinant DNA techniques offers many possibilities to create capable systems. This chapter describes the most important whole cell biotransformations developed in the past as well as relevant processes with small-scale and technical application. 

Several aspects have to be considered in order to regenerate NAD(P)H by an indirect electrochemical procedure without the application of a second regeneration enzyme The active redox catalyst should transfer two electrons in one step or a hydride ion. At potentials more negative than —0.9 V vs SCE, NAD+ dimers will be formed, so the electrochemical activation of the catalyst should be possible at potentials less negative than —0.9 V. To prevent low chemoselectivity and low enantioselectivity, the active form of the catalyst should not transfer the electrons or the hydride ion directly to the substrate but to NAD(P)+. Furthermore, only active 1,4-NAD(P)H should be formed [90]. 

Wild type cells often contain several enzymes which carry out the same reaction. Unfortunately, in many cases these enzymes produce compounds with opposite stereoselectivity. Therefore, whole cells in which such enzymes are present cannot be applied for the synthesis of enantiomerically pure products. To increase the stereoselectivity of a whole cell reaction, recombinant DNA techniques need to be applied. Very common is the overexpression of the enzyme, which catalyzes the particular reaction in a suitable heterologous host such as E. coli. The simultaneous overexpression of an enzyme which catalyzes the regeneration of the consumed cofactor is highly efficient. Ideally, growing cells should provide simultaneously the enzyme for the desired reaction as well as the cofactor regenerating enzyme. Such so-called designer cells seem to be very promising for technical applications. 

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. 

---