Engineered cationic methacrylated chitosan hydrogels as multifunctional biocompatible platforms for robust tyrosinase immobilization

Despite the broad potential of tyrosinase in biocatalysis, its practical application remains limited by poor operational stability and restricted reusability. Here, we developed two engineered cationic methacrylated chitosan-based hydrogels as biocompatible platforms for immobilization of highly purified Agaricus bisporus tyrosinase. The enzyme was purified to near homogeneity through sequential gel filtration and ion-exchange chromatography and subsequently immobilized using a combined adsorption-entrapment strategy within positively charged hydrogel networks. The cationic matrices were designed considering the net negative surface charge of tyrosinase to enhance enzyme retention while physically entrapping the enzyme within the hydrogel network. The physicochemical properties of the hydrogels were characterized before and after enzyme immobilization to assess the impact of enzyme incorporation. Both hydrogel formulations exhibited enzyme loading capacities of 42.02 and 39.37 μL g⁻ 1 , respectively, while retaining 94.56 and 90.46% of the free enzyme’s diphenolase activity, respectively. Comparative evaluation identified one formulation as the optimal hydrogel, exhibiting superior overall catalytic performance, operational stability, and storage stability, and was therefore selected for further biological characterization. Kinetic analysis revealed negligible changes in substrate affinity together with approximately 90–95% retention of the maximum catalytic activity (V max ), indicating preservation of the enzyme's catalytic function after immobilization. Compared with the free enzyme, the optimized immobilized tyrosinase exhibited a shift in the optimum pH from 6.5 to 5.5 and an increase in the optimum temperature from 30 to 50 °C, retained 81% of its initial activity after five consecutive catalytic cycles, and maintained approximately 82% residual activity after 28 days of storage at 4 °C. Furthermore, the optimized hydrogel demonstrated pronounced antibacterial activity against both Escherichia coli and Staphylococcus aureus while exhibiting negligible cytotoxicity and hemolytic activity, confirming its high biocompatibility. Collectively, these findings establish engineered cationic methacrylated chitosan hydrogels as multifunctional and biocompatible platforms for tyrosinase immobilization, providing high immobilization efficiency, enhanced operational stability, antibacterial activity, and favorable biological compatibility, with promising potential for industrial biocatalysis and biomedical applications.