Substrate-coupled cofactor

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

The use of organic solvents as reaction media for biocatalytic reactions can not only overcome the substrate solubility issue, but also facilitate the recovery of products and biocatalysts as well. This technique has been widely employed in the case of lipases, but scarcely applied for biocatalytic reduction processes, due to the rapid inactivation and poor stability of redox enzymes in organic solvents. Furthermore, all the advantages for nonaqueous biocatalysis can take effect only if the problem of cofactor dependence is also solved. Thus, bioreductions in micro- or nonaqueous organic media are generally restricted to those with substrate-coupled cofactor regeneration. 

Substrate-Coupled Cofactor Regeneration with Recombinant Whole Cells... 

Enzymatic cofactor regeneration can be subdivided into two categories the enzyme-coupled approach, where two different enzymes are used (one for the production reaction, and one for the regeneration reaction) and the substrate-coupled approach, where one and the same enzyme is used for both production and regeneration (E = E2). The most convenient and commonly used enzymatic regeneration systems are summarized in Table 43.1. 

In the second approach the reducing equivalents are suppHed by a nicotinamide cofactor (NADH or NADPH) and for commercial viability it is necessary to regenerate the cofactor using a sacrificial reductant ]12]. This can be achieved in two ways substrate coupled or enzyme coupled (Scheme 6.2). Substrate-coupled regeneration involves the use of a second alcohol (e.g. isopropanol) that can be accommodated by the KRED in the oxidative mode. A problem with this approach is that it affords an equilibrium mixture of the two alcohols and two ketones. In order to obtain a high yield of the desired alcohol product a large excess of the sacrificial alcohol needs to be added and/or the ketone product (acetone) removed... 

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.

The first approach requires a second enzyme as well as a second substrate for regeneration. In the second method, the substrate-coupled regeneration, one single enzyme is responsible for the formation of the desired product as well as for cofactor recycling. 

Fig. 2 Different principles for the regeneration of nicotinamide cofactors. Method 1 describes the regeneration using a second enzyme, method 2 shows the substrate-coupled approach utilizing one enzyme for the main reaction, the reduction of the substrate as well as for the regeneration of NAD(P)H...

Coupled-Enzyme Approach. The use of two independent enzymes is more advantageous (Scheme 2.112). In this case, the two parallel redox reactions - i.e., conversion of the main substrate plus cofactor recycling - are catalyzed by two different enzymes [721]. To achieve optimal results, both of the enzymes should have sufficiently different specificities for their respective substrates whereupon the two enzymatic reactions can proceed independently from each other and, as a consequence, both the substrate and the auxiliary substrate do not have to compete for the active site of a single enzyme, but are efficiently converted by the two biocatalysts independently. 

Only in the case of alcohol dehydrogenase (ADH)-catalyzed reactions, a substrate-coupled system can be applied, in which the enzyme that transforms the substrate of interest also regenerates the cofactor at the expense of a co-substrate to be used in at least stoichiometric amounts with respect to the substrate. 

As previously mentioned, an ADH can be used for the in situ recycling of NAD(P)H cofactors by exploiting the so-called substrate-coupled approach, that is, the coupHng of the reaction of interest with a secondary reaction running in the reverse direction... 

Alcohol dehydrogenase-catalyzed regeneration of NAD(P)H by oxidation of alcohols certainly represents the most widespread regeneration method currently applied. Especially if the desired production reaction is an ADHsubstrate-coupled regeneration approach excels in simplicity, as only one biocatalyst has to be used for the whole reaction (Scheme 8.8). Another advantage of this methodology is that the nicotinamide cofactor does not have to leave the... 

Isopropanol can be used in the asymmetric hydrogen transfer reactions for two purposes first, as a cosolvent for improving the substrate solubility and second, as a cosubstrate for cofactor regeneration (substrate-coupled approach). For example, in the reduction of acetophenone by Candida viswanathii cells, addition of 10% (v/v) isopropanol led to a great increase of the substrate tolerance and a conversion of 90% compared to 9% in the control at a substrate concentration of 70 mM after lh[8]. 

Figure 9.4 Biocatalytic reduction of acetophenone and 3-butyn-2-one using lyophilized coli cells with overexpressed carbonyl reductase in neat substrates with isopropanol-coupled cofactor regeneration.

Cofactor regeneration is potentially possible via electrochemical, chemical, photochemical, and enzymatic methods. The enzymatic methods are of two types those that are enzyme-coupled (using two enzymes) and those that are substrate-coupled. Table 10.3 lists some of the regeneration options. The substrate-coupled approach is particularly attractive [32]. In such systems, it is common to use 2-propanol [isopropyl alcohol (IPA)] as the cosubstrate that leads to acetone as the coproduct. The complication with such a system is that a competitive equilibrium is established... 

Besides wild-type strains, more recently the use of recombinant whole cells has gained increasing popularity for application in asymmetric ketone reduction. When overexpressing the ADFi only, in situ cofactor recycling based on a "substrate-coupled approach" represents a favorite approach as demonstrated in an early contribution by the Itoh group [86] utilizing a recombinant ADH from a Corynebacterium overexpressed in E. coli. This concept has been also applied by Daicel researchers in the presence of an E. coli catalyst with recombinant ADH from Candida parapsilosis. This biocatalyst catalyzes the reduction of p-ketoester 28 at a 36.6 g/1 substrate loading and fimiished the alcohol (R)-29 in 95.2% yield and with 99%ee (Scheme 23.12) [87]. 

As selected examples of more recently developed ketone reductions with recombinant whole cells following the concept of substrate-coupled in situ cofactor recycling, the work by the Schmid and Buehler group with a recombinant ADH from Thermus sp. [88] and by the Kroutil group with a DMSO-tolerant recombinant ADH from Paracoccus pantotrophus [89] shall be mentioned here. This recombinant whole-cell... 

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