Biocatalyst substrate-coupled cofactor

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. 

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. 

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... 

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]. 

Single diastereomers of the diol products were obtained by a two-step process after careful selection of the biocatalysts. In the first reduction step, the starting substrate was completely converted into the corresponding P-hydroxy ketone by the first KRED enzyme without any further reduction to the 1,3-diol. Reduction reactions were coupled with the glucose/glucose dehydrogenase (GDH) system for the in situ regeneration of the reduced cofactor. The second KRED enzyme was then added to... 

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