Complex Coacervate Emulsions as a Strategy to Stabilize Enzymes for Catalysis in Organic Solvents

Complex coacervates have emerged as versatile platforms for protein encapsulation, enabling enzymatic catalysis in aqueous environments. Despite their potential, applications of coacervates are limited by the substrate solubility in water. In this study, we present a protocol to stabilize enzyme-loaded coacervate droplets in water-immiscible organic solvents via the formation of highly stable emulsions. These emulsions were formed using coacervates composed of poly(diallyldimethylammonium hydroxide) and poly(acrylic acid), stabilized by a polystyrene-based, amphiphilic, anionic copolymer in toluene, chlorobenzene, chloroform, and dichloromethane. The resulting microdroplets display exceptional resistance to coalescence, including after centrifugation, and remain stable for weeks. This stability facilitates their separation and redispersion for use in repeated catalytic applications. Using α-chymotrypsin as a model enzyme, we show that the aqueous microenvironment within the droplets maintains enzyme stability over time and enables biocatalysis in nonaqueous media.