Enzyme‐Polymer‐Conjugate‐Based Pickering Emulsions for Cell‐Free Expression and Cascade Biotransformation

Abstract In this study, we addressed the limitations of conventional enzyme‐polymer‐conjugate‐based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from nonspecific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non‐canonical amino acid (ncAA) at a specific site on target enzymes, we enabled precise polymer‐enzyme conjugation. These engineered conjugates then acted as biocatalytically active emulsifiers to stabilize Pickering emulsions, while encapsulating a cell‐free protein synthesis (CFPS) system in the aqueous phase for targeted enzyme expression. The resulting cascade reaction system leveraged enzymes expressed in the aqueous phase and on the emulsion interface for optimized chemical biosynthesis. The use of the cell‐free system eliminated the need for intact whole cells or purified enzymes, representing a significant advancement in biocatalysis. Remarkably, the integration of Pickering emulsion, precise enzyme‐polymer conjugation, and CFPS resulted in a fivefold enhancement in catalytic performance as compared to traditional single‐phase reactions. Therefore, our approach harnesses the combined strengths of advanced biochemical engineering techniques, offering an efficient and practical solution for the synthesis of value‐added chemicals in various biocatalysis and biotransformation applications.