Effect of pH and Particle Charge on the Interfacial Properties of Biocatalytic Pickering Emulsions─Where Are the Enzymes Located?

Pickering emulsions (PEs), where water-in-oil (w/o) droplets are stabilized by nanoparticles (NPs), offer a promising platform for biocatalysis by providing a large interfacial area crucial for efficient substrate conversion. While several lipase catalyzed reactions in PEs have been demonstrated, the exact interfacial structure is unknown. This study focuses on the interfacial network formed by NPs and <i>Candida rugosa</i> lipase (CRL) at the octanol/water-interface by varying pH and NP charge. By applying different methods, the location of lipases within a PE was identified and the enzyme concentration profile quantified for the first time. Positively charged nanoparticles (NP+) adsorbed at the o/w-interface together with CRL to form a network-structure. The relation between individual and simultaneous adsorption showed a constant value of 0.75 for the investigated pH range. Negatively charged particles (NP-) did not adsorb spontaneously at the negatively charged octanol/water-interface and therefore showed no influence on the enzyme adsorption behavior. Interfacial shear rheology measurements further revealed distinct elastic behavior of the enzyme-particle network due to attractive interactions between positively charged nanoparticles and CRL. This was shown by a 4.4-fold increase in the interfacial storage modulus. In contrast, repulsive interactions─either between CRL and positively charged particles at low pH or with negatively charged particles─did not enhance the elastic response of CRL at the interface. Confocal laser scanning microscopy of prepared PE droplets showed an interfacial CRL layer thickness of 0.75 μm for NP+ and 0.51 μm for NP-. Using NP+ results in a 30% higher interfacial enzyme concentration, indicating a more compact layer structure. These insights contribute to optimizing biocatalytic systems using PEs for industrial applications and provide a basis for the quantitative analysis of the interfacial layer in a Pickering emulsion.