Biocatalyst enzyme-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. 

The electrochemical sensing of NADH is of great interest in the development of a dehydrogenase-based amperometric biosensor owing to the ubiquitous use of NADH as a cofactor for over 300 enzymes and in the fine chemicals industry using NAD -dependent biocatalysts [106]. The oxidation of NADH at bare and modified electrodes has been well studied and the oxidation process is dependent on the nature of the electrode used. The direct electrochemical oxidation of NADH at the bare electrode, irrespective of its nature, requires a high overpotential, despite the formal potential of the NAD /NADH redox couple at pH 7, which is reported to be... 

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. 

In fact, successful deracemization was not achieved when using a cell-free extract of the Alcaligenes cells, which showed NADH-dependent (R)-selective ADHs active in the oxidation reaction, coupled to a NADH-dependent (S)-selective ADH for the reduction reaction. Instead, it was easily achieved when using microbial whole cells in the biooxidation reaction or when combining the same cell-free extract with a NADPH-dependent ADH. In both cases, possible short circuits between the two cofactor regeneration systems were therefore avoided, thanks to the compartmen-talization of one of the involved biocatalysts in the cells or to the use of enzymes with different cofactor specificity. 

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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