Regioselective Lipase Anchoring on Janus Nanoparticles Enables Directional Oil–Water Interfacial Assembly in Pickering Emulsions for Enhanced Biocatalysis

ABSTRACT Precise control over enzyme structure through carrier design is key for high performance biocatalysis. However, achieving selective enzyme orientation and creating a defined local interface environment remain challenging. In this work, amphiphilic dumbbell‐shaped Janus polymer nanoparticles (DS‐JPNs) were designed to anchor Aspergillus niger lipase (ANL) on one side, ensuring the catalytic domain faces outward. The distinct hydrophilic and hydrophobic lobes create different environments that stabilize the enzyme and help open its lid during assembly at interfaces. These DS‐JPN@ANL particles were used as a solid stabilizer in Pickering emulsions. The anisotropic DS‐JPNs stabilize the interface with a large surface area that improves mass transfer while preserving enzyme activity. Confocal microscopy and fluorescence spectroscopy showed that this directional design activates the enzyme at the interface without altering its native structure. As a result, DS‐JPN‐immobilized ANL achieves a 239‐fold higher initial turnover rate than free ANL and retains over 87% activity after five cycles, producing about 89% fatty acid methyl ester (FAME) yield under mild conditions. This study establishes a clear structure–function relationship between Janus carrier anisotropy, directional Pickering emulsion assembly and lipase activation, offering a general strategy for constructing interfacial biocatalysts with controlled orientation and tailored microenvironments.