Solvent Effects in Bioreductions

Over the past three decades, an increasing concern was put on application of nonaqueous solvents to facilitate biocatalytic reactions where several industrially attractive advantages are presented, such as increased solubility of nonpolar substrates, reversal of hydrolysis reactions, alternation of enzyme selectivity, and suppression of water-dependent side reactions. However, there are some inherent problems and technical challenges, including inactivation of biocatalysts, potentially reduced protein stability and lowered reaction rates due to mass-transfer limitations, and/or the increased rigidity of protein structure. 

Numerous studies have revealed that biocatalytic reductions can be performed well in the reaction media containing nonaqueous solvents, which vary from conventional organic solvents to greener solvents, such as ionic liquids and supercritical fluids. The use of nonaqueous solvents has not only enhanced the efficiency of bioreductions by allowing the reactions to be conducted at high substrate concentrations, but also altered enzymatic selectivity, including chemo-, regio-and enantioselectivities. 

In this chapter, latest advancements in solvent engineering in bioreductions and greener needs for bioreaction media have been discussed in depth with recent examples. Solvents for bioreductions may be categorized as (i) aqueous (ii) water/water-miscible (monophasic aqueous-organic system) (iii) water/ water-immiscible (biphasic aqueous-organic system) (iv) nonaqueous (mono-phasic organic system, including solvent-free system) and (v) nonconventional media (e.g., ionic liquids, supercritical fluids, gas-phase media, and reverse micelles). 

Synthetic Methods for Bido cally Active Molecules Exploring the Potential of Bioreductions, First Edition. Edited by Elisabetta Brerma- 

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In many cases, the substrates for reaction are poorly water-soluble, meaning it is necessary to consider organic solvent or two-liquid-phase systems to ensure the concentration of substrate (and hence product) in the reactor is sufficient. Two-liquid-phase systems may also be used for product removal as discussed later. There are relatively few reports of solvent effects on the various bioreduction enzymes, but a recent publication found ene-reductases to be robust in several aqueous-organic two-phase systems [9]. 

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. 

Numerous bioreduction examples use an organic solvent to form a second phase from extractive ISPR. For example, extractive ISPR in a two-phase system was used to remove 2-phenylethanol, giving an increase of one order of magnitude in product concentration and also productivity [41]. The two-phase system was ultimately limited by mass transfer on account of high viscosity of the chosen organic phase (oleic acid). In common with many such processes, it is clear that far more work is required on solvent selection. Interestingly the dissolved level of solvent in water is always exposed to the biocatalyst (whether the ISPR is internal or external) and in many cases this causes a detrimental effect to the biocatalyst over time. 

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