Dehydrogenases enzyme-coupled regeneration

R)-2-Hydroxy-4-phenylbutyric acid was produced continuously in an enzyme membrane reactor by enzymatic reductive animation of the a-keto acid with d-lactate dehydrogenase coupled with formate dehydrogenase (FDH) for regeneration of NADH. Reactor performance data matched a kinetic reactor model (Schmidt, 1992). 

Figure 19.5. Cofactor regeneration systems for NAD(P)H-dependent enzyme reactions. The enzyme-coupled one involving GDH (a), that involving FDH (b), and the substrate-coupled one (c). AR1, aldehyde reductase from S. salmonicolor Leu DH, leucine dehydrogenase ADH, sec-alcohol dehydrogenase.

Various concepts for the enzymatic regeneration of NAD4 in combination with isolated HLADH have been reported, ranging from a second dehydrogenase such as glutamate dehydrogenase151, S21 to enzyme-coupled or intrasequential approaches. 

For example, horse liver alcohol dehydrogenase (HLADH) was noncovalently immobihzed on a membrane and packed into a PBR [74] operated in a recirculated loop mode for the reduction of racemic 2-phenyl-tetrahydropyran-4-one 1 in the presence of NADH. The HLADH-reactor coupled with an enzymic cofactor regeneration system in the mobile phase could convert the substrate to the enantiopure (S,S)- and (R,S)-2. The immobilized HLADH reactor was stable over 6 months when stored at 5 °C. 

T Eguchi, T lizuka, T Kagotani, JH Lee, I Urabe, H Okada. Covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermopMlus malate dehydrogenase NAD(H)-regeneration unit for a coupled second-enzyme reaction. Eur J Biochem 155 415 21, 1986. 

The importance of having adequate supplies of NADPH for the regeneration of these various enzymes cannot be over emphasized. In normal situations this cofactor can be adequately provided by the reductive pentose phosphate pathway. Monitoring the activity of the pentose phosphate pathway has been proposed as a unique way to study the metabolic response to oxidative stress, since the glutathione peroxidase activity is coupled via glutathione reductase to the enzyme glucose-6-phosphate dehydrogenase (Ben Yoseph et ah, 1994). 

It is difficult to incorporate dehydrogenases that are coupled with NAD(P) into amperometric enzyme sensors owing to the irreversible electrochemical reaction of NAD. We have developed an amperometric dehydrogenase sensor for ethanol in which NAD is electrochemically regenerated within a membrane matrix. 

In contrast, amino acid dehydrogenases comprise a well-known class of enzymes with industrial apphcations. An illustrative example is the Evonik (formerly Degussa) process for the synthesis of (S)-tert-leucine by reductive amination of trimethyl pyruvic acid (Scheme 6.12) [27]. The NADH cofactor is regenerated by coupling the reductive amination with FDH-catalyzed reduction of formate, which is added as the ammonium salt. 

Table 16. Chiral alcohols produced by continuous enzyme-catalyzed processes. The corresponding ketones are reduced with (S)-ADH from Rhodococcus erythropolis, NADH was regenerated by simultaneous coupling with formate dehydrogenase from Candida boidinii (FDH) and formate (data from [159])...

Fig. 8 Synthesis of amino acids by a multienzyme system consisting of leucine dehydrogenase (LeuDH) catalyzing the reductive amination of the corresponding keto acid, L-lactate dehydrogenase (l-LDH), and lactate for the regeneration of NADH and urease for the in situ generation of ammonia. The coenzyme NAD+ was covalently bond to dextran, enzymes and dextran-coupled NAD+ were...

Association between enzymatic and electrochemical reactions has provided efficient tools not only for analytical but also for synthetic purposes. In the latter field, the possibilities of enzymatic electrocatalysis, e.g., the coupling of glucose oxidation (catalyzed either by glucose oxidase or glucose dehydrogenase) to the electrochemical regeneration of a co-substrate (benzoquinone or NAD+) have been demonstrated [171, 172]. An electroenzymatic reactor has also been developed ]172] to demonstrate how the enzyme-electrode association can be used to produce biochemicals on a laboratory scale. 

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