pH-Driven Enzymatic Breakdown and Release of Catalase from Alginate Hydrogel

Stimuli-induced release resulting in biochemical transformations has received a lot of attention due to its application in controlled drug release. In this work, catalase (EC 1.11.1.6) and trypsin (EC 3.4.21.4) were simultaneously encapsulated into a pH-responsive alginate hydrogel. Upon applying electrochemical potential -0.8 V vs Ag/AgCl/KCl resulting in oxygen reduction, which generates a local pH increase, trypsin becomes active. The activated trypsin provides the digestion of catalase within the alginate matrix, stimulating the release of active subunits. Simultaneously with the trypsinolisis of catalase, the pH increase led to hydrogel swelling, allowing for the release of catalase active fragments. Difference in the release behavior was also observed in solutions with different bulk pH values, at which trypsin was or was not active. Labeling of catalase with rhodamine B isothiocyanate was performed for the release observation using confocal fluorescence microscopy and regular fluorescent spectroscopy. The activity of catalase fragments was analyzed using a UV-visible spectrophotometer, following the enzymatic assay toward guaiacol, which is known to be a selective substrate for catalase subunits. Blue native polyacrylamide gel electrophoresis was used to analyze the efficiency of trypsinolysis and the molecular weights of the formed fragments. The proposed signal-stimulated release of bioactive fragments from the alginate hydrogel presents an intriguing model system with the potential for biomedical applications.