Co-immobilization of Enzymes and Cofactors Enabled by Liquid-Liquid Phase Separation for Continuous-Flow Catalysis

Abstract

Co-immobilization of enzymes and cofactors for continuous-flow catalysis faces significant challenges in maintaining the accessibility and activity of immobilized components. Herein, we design a liquid-liquid phase separation (LLPS) system incorporating long-chain polyelectrolytes, which enables efficient co-immobilization of enzymes and cofactors via the electrostatic interaction and the macromolecular crowding effect. Remarkably, the total turnover number (TTN) of cofactors reached a maximum of 1510, which outperforms most continuous-flow systems. Our system, requiring no solid carriers and chemical band modifications, provides a versatile platform for constructing self-sufficient biocatalytic processes with high activity and stability.