Kadir Erol, Hüseyin Alkan, İhsan Alacabey
Cryogels with interconnected macroporous architectures offer significant advantages as enzyme immobilization supports due to their high permeability, mechanical robustness, and tunable surface chemistry. In this study, a novel Poly(HEMA-co-GMA) cryogel was synthesized and subsequently modified through polyethyleneimine (PEI) grafting and transition-metal chelation to create high-affinity matrices for catalase immobilization. Among the metal ions tested with Cu(II), Ni(II), and Co(II), the Cu(II)-functionalized cryogel exhibited superior physicochemical properties, including the highest water retention capacity (438.4%), well-preserved porosity, and strong coordination interactions with amine-rich PEI domains. FT-IR, SEM, TGA, BET, elemental analysis, and ICP-OES results confirmed successful stepwise modification and metal loading. Catalase immobilization studies revealed that the Poly(HEMA-co-GMA)-PEI-Cu(II) cryogel achieved the highest enzyme loading (391.9 mg·g⁻¹), with an optimal immobilization time of 8 h and optimum pH near neutrality. Kinetic analysis demonstrated a substantial decrease in Km (from 57.3 to 14.4 mM), indicating enhanced substrate affinity, while kcat/Km increased 2.8-fold relative to the free enzyme. The immobilized catalase exhibited improved thermal tolerance, strong operational stability (34.2% activity after 15 cycles), high desorption efficiency (96% in the first cycle), and markedly superior storage stability (62.1% activity after 70 days at 4 °C) compared to its free counterpart. These results validate the Cu(II)-chelated Poly(HEMA-co-GMA)-PEI cryogel as a highly efficient and reusable biocatalytic platform with significant potential for industrial and environmental enzyme-based applications.