Encapsulation of multiple enzymes within a microgel via water-in-water emulsions for enzymatic cascade reactions.

Enzyme-loaded spherical microgels with diameters of several micrometers have been explored for use in therapeutic microreactors and biosensors. Conventional preparation strategies for enzyme-loaded microgels utilized water-in-oil emulsions or flow chemistry techniques. The former damage enzyme activity using organic solvents and the latter are expensive and difficult to expand because of the complex system. In this study, we present a simple strategy for creating multiple enzyme-loaded gelatin-based microgels with tunable diameters in a single flask. This strategy was based on our finding that enzymes spontaneously partitioned in a dispersed methacryloyl gelatin aqueous solution in a poly(vinylpyrrolidone) (W GelMA /W PVP ) aqueous solution. The method achieved an encapsulation efficiency of over 70% even with four types of enzymes and retained their activity owing to the full aqueous system. Additionally, the encapsulated β-galactosidase activity was maintained for 24 hours at pH 6, although naked β-galactosidase lost approximately 60% of its activity, which was superior to that of previous enzyme-loaded gelatin gels. Moreover, this simple method enabled the production of 10 g-scale or more microgels in one batch. We also demonstrated that multiple enzyme-loaded gelatin microgels functioned as cascade microreactors for lactose and glucose sensing. This versatile strategy enables the production of enzyme-loaded microgels while maintaining the enzyme activity using very low technologies. This result contributes to the easy preparation of enzyme-loaded microgels and their applications in the biomedical and green catalytic fields.