Design of fusion enzymes for biocatalytic applications in aqueous and non-aqueous media

Abstract

Biocatalytic cascades play a fundamental role in sustainable chemical synthesis. Fusion enzymes are one of the powerful toolboxes to enable the tailored combination of multiple enzymes for efficient cooperative cascades. Especially, this approach offers a substantial potential for the practical application of cofactor-dependent oxidoreductases by forming cofactor self-sufficient cascades. Adequate cofactor recycling while keeping the oxidized/reduced cofactor in a confined microenvironment benefits from the fusion fashion and makes the use of oxidoreductases in harsh non-aqueous media practical. In this mini-review, we have summarized the application of various fusion enzymes in aqueous and non-aqueous media with a focus on the discussion of linker design within oxidoreductases. The design and properties of the reported linkers have been reviewed in detail. Besides, the substrate loadings in these studies have been listed to showcase one of the key limitations (low solubility of hydrophobic substrates) of aqueous biocatalysis when it comes to efficiency and economic feasibility. Therefore, a straightforward strategy of applying non-aqueous media has been briefly discussed while the potential of using the fusion oxidoreductase of interest in organic media was highlighted.

CYP116B5‐SOX: An artificial peroxygenase for drug metabolites production and bioremediation

“…[ 43 ] As the research in the field of fusion enzymes expanded extensively in the last few years, further possible optimization of protein engineering emerged, including the modification of the sequence of the linker in terms of length and/or rigidity, as well as use other H 2 O 2 ‐generation system(s). [ 84,85 ] Interestingly, the Molecular Lego approach we pioneered, allow us to modify each module of the chimera—, that is, the linker sequence or the H 2 O 2 ‐donor domain—to further optimize the stability and activity of the P450 fusion enzyme. In light of the previous results, herein the fusion enzyme has been heterologously expressed in E. coli , purified and characterized in terms of domain functionalities, thermal stability and tolerance toward H 2 O 2 (Figures 2, 3, and 4).…”

Bicyclo[3.2.0]carbocyclic Molecules and Redox Biotransformations: The Evolution of Closed-Loop Artificial Linear Biocatalytic Cascades and Related Redox-Neutral Systems

“…Careful design of such chimeric bifunctional enzymes proved to be challenging in order to both optimise stability and promote effective substrate and cofactor channeling within the chimeric complexes. These developments have been reviewed comprehensively in context by Aalbers and Fraaije [281], and have continued to be progressively updated [282].…”

Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media

“…The fusion form of enzymes could shorten the transport distance of cofactors while diminishing the impact of organic solvents on unstable nicotinamide cofactors. Additionally, it can enhance the kinetics by faster provision of the reduced or oxidized nicotinamide cofactors [30–32] . Exemplarily, Huang et al [31] .…”

“…Additionally, it can enhance the kinetics by faster provision of the reduced or oxidized nicotinamide cofactors. [30][31][32] Exemplarily, Huang et al [31] reported the first use of fused type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) in microaqueous media (5 vol. % water).…”