Combining Protein Phase Separation and Bio‐orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn²⁺ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.