Beneficial substrate partitioning boosts non-aqueous catalysis in de novo enzyme-alginate beads

Synthetic reactions often require solvents incompatible with biocatalysts. Here, we encapsulate a de novo heme-containing enzyme, C45, in calcium-alginate hydrogel beads to facilitate heterogeneous biocatalysis in neat organic solvents. Post-encapsulation, C45 retains activity even when the beads are suspended in organic solvents. In particular, the carbene transferase activity of C45 is enhanced when reactions are performed in aprotic, non-polar solvents such as hexane and toluene. Activity-solvent dependencies reveal that this activity boost is likely due to beneficial partitioning of the substrate into the beads from the organic phase. Furthermore, encapsulation facilitates enzyme recovery and recycling after the reaction. Such encapsulation opens up novel opportunities for biocatalysis in organic solvent systems, combining desired solvent properties of organic chemistry with enzymatic selectivity and proficiency.