Engineering Polyethylenimine-Metal Functionalized Cryogels for Superior Catalase Binding, Activity, and Long-Term Durability

Abstract

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 & centerdot;g(-)& sup1;), with an optimal immobilization time of 8 h and optimum pH near neutrality. Kinetic analysis demonstrated a substantial decrease in K-m (from 57.3 to 14.4 mM), indicating enhanced substrate affinity, while k(cat)/K-m 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 degrees 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.