Biocatalysis cofactor recycling

An important technical issue is the large-scale applicability of co-factor-dependent enzymatic systems. It is generally accepted that, e.g., NADH-requiring oxidoreductases can easily be used in whole-cell biocatalysis such as baker s yeast-mediated reductions, where the cofactor recycling step is simultaneously performed within the intact cell, driven by the reduction equivalents introduced via the external carbon and energy source (glucose). 

The whole-cell biocatalysis approach is typically used when a specific biotransformation requires multiple enzymes or when it is difficult to isolate the enzyme. A whole-cell system has an advantage over isolated enzymes in that it is not necessary to recycle the cofactors (nonprotein components involved in enzyme catalysis). In addition, it can carry out selective synthesis using cheap and abundant raw materials such as cornstarches. However, whole-cell systems require expensive equipment and tedious work-up because of large volumes, and have low productivity. More importantly, uncontrolled metabolic processes may result in undesirable side reactions during cell growth. The accumulation of these undesirable products as well as desirable products may be toxic to the cell, and these products can be difficult to separate from the rest of the cell culture. Another drawback to whole-cell systems is that the cell membrane may act as a mass transport barrier between the substrates and the enzymes. 

Within biocatalysis, redox reactions especially are often carried out in biphasic systems using crude cell systems, as the recycling of expensive cofactors is vital. The organic phase, which holds the rather insoluble substrate (and products), can be replaced by a more harmless IL. By this means, baker s yeast was used by Howarth and co-workers for enantioselective reduction of ketones [37]. In a 10 1 mixture of [BMIM][PF6] and water, reactions proceeded for a range of substrates giving yields and selectivities very comparable to those obtained by conventional methods [Eq. (8)]. 

SAM and related cofactors, isolated or produced by total synthesis, are highly priced and their stoichiometric consumption requires one or even more equivalents. An economically feasible cofactor-dependent biocatalysis thus rehes on the substoichiometric or even catalytic use of the cofactor associated with a cofactor regeneration system, ideally in a cascaded reaction sequence. Such enzymatic conversions are assisted by an additional recycling reaction which restores the... 

Solid-gas biocatalysis has not been restricted to the use of isolated enzymes. Whole cells are of particular interest where the in vivo recycling of cofactor can be achieved by addition of a cosubstrate. Dried Saccharomyces cerevisiae cells have been used to catalyze the continuous reduction of hexanal to hexanol with in situ regeneration of NADH via oxidation of ethanol in a solid-gas system [81]. The... 

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