Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Abstract

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

Engineering stereoselectivity of ThDP-dependent enzymes

“…The same trend was observed with acetoin, which is formed with an ee of 85% ( S ) in buffer but with only 28% ee ( S ) in the presence of 30 vol% ethanol. This work demonstrated the potential of solvent engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations .…”

Enzyme–Polymer Conjugates as Robust Pickering Interfacial Biocatalysts for Efficient Biotransformations and One‐Pot Cascade Reactions

“…Benzaldehyde lyase (BAL) was chosen as amodel enzyme for conjugate formation and interfacial catalysis for two reasons:1 )BAL is av ulnerable enzyme that is quickly deactivated at aliquid-liquid interface, [16] and its use can thus verify whether polymer conjugation improves enzyme stabil-ity at interfaces;2 )its catalytic center is not surrounded/ associated with lysine residues,which are the optimal sites for grafting ahigh number of polymer chains to the enzyme (see Figures S1 and S2 in the Supporting Information).…”

Performance of Recombinant‐Whole‐Cell‐Catalyzed Reductions in Deep‐Eutectic‐Solvent–Aqueous‐Media Mixtures

“…Results are depicted in Figure 5. dependent lyases and co-solvents, [20] as well as when ionic liquids are added as performance additives. [8a] In any case, the use of non-hazardous DES offers not only better outcomes (higher stereoselectivities), but also more environmentally-friendly conditions for biocatalysis using recombinant whole-cells.…”