Bioreduction cofactor regeneration

A more efficient and simple bioreduction system, which avoids the problems with the system involving naturally-occurring microorganisms or cell-free systems described above, has been required. A novel bioreduction system, in which Esherichia coli transformant cells coexpressing the genes of an NAD(P)H-dependent carbonyl reductase and GDH, as a cofactor regenerator, were used as the catalyst, has been constructed (Kataoka et al., 2003). The production of chiral CHBEs is a typical successful example with this bioreduction system. 

Figure 19.6. Multi-purpose bioreduction system, involving recombinant microorganisms coexpressing carbonyl reductase and cofactor regenerator genes.

One of the prominent industrial bioreduction processes, run by cofactor regeneration, is performed by leucine dehydrogenase. This enzyme can catalyze the reductive amination of trimethylpyruvic acid, using ammonia (see Fig. 3.45). For this process the cofactor regeneration takes place by using formate as the reductant and formate dehydrogenase as the second enzyme. The advantage of... 

In this chapter we would like to highlight some recent examples, from 2006 onward, regarding the employment of whole-cell systems or (partially) purified enzymes to obtain, through bioreduction, biologically relevant (family of) compounds or intermediates, focusing on the enzymatic preparation and the cofactor regeneration system employed (Scheme 4.1). This chapter has been divided depending on the chemical structure of the substrates. Thus, Section 4.2 will provide recent examples of bioreductions over a- or P-keto esters (Section 4.2.1), diketones (Section 4.2.2), halo... 

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. 

Toxic substrates and products to whole-cell biocatalysts. Finally, in whole-cell format, the substrate and/or product of the bioreduction can be toxic to the cells, preventing cofactor regeneration. Such irreversible loss of regeneration capacity is, of course, catastrophic for the process. In principle, this can be overcome by maintaining a low substrate concentration, but this will ultimately prevent a sufficiently high product concentration for an effective process. In some cases, dependent upon the water-solubility (and if the substrate is a liquid), it may be possible to feed the substrate, such that a low concentration is provided to the cells in the reactor, but at the end of the reaction a high product concentration is achieved. However, in nearly all cases at the required concentration for an... 

For the aforementioned reasons the use of isolated enzymes is often preferred due to reduction in side reactions and higher productivities (see Ref. [12] for a review of this topic). However this brings other challenges such as the need for effective cofactor regeneration. The choice between enzyme and whole-cell biocatalysts is complex and requires more work in the future to establish a clearer strategy to help the process design and implementation of bioreductions. 

What are the bottelnecks for bioreduction The drawbacks of a bioreduction process involving whole cells of microoganisms can be summarized i) Microbial strains possessing both carbonyl reductase activity and cofactor (NAD(P)H)-regenerating activity are necessary to obtain a highmolar yield, because a stoichiometric amount of cofactor is required for substrate reduc-... 

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