Abstract
Although catalase is a crucial antioxidant enzyme widely used in industrial and environmental systems to decompose hydrogen peroxide (H2O2), its low stability and reusability limit its practical applications. This study aimed to enhance catalase performance by immobilising an enzyme on titanium dioxide nanoparticles (TNPs) in the anatase phase to form catalase-immobilised TNPs (CITNPs) and to evaluate their catalytic activity, stability, and reusability under various conditions. TNPs were modified using four chemical intermediates to introduce functional groups for enzyme immobilisation: (3-aminopropyl)triethoxysilane for amine groups (method A), (3-mercaptopropyl)trimethoxysilane for thiol groups (method B), dihydrocaffeic acid for carboxylic groups (method C), and tannic acid for phenolic groups (method D). Catalase was immobilised onto each modified TiO2 intermediate to obtain CITNPs. The catalase activity, thermal and pH stabilities, reusability, and functional capability in beverage solutions were evaluated using ultraviolet-visible analyses and enzyme activity assays. All four immobilisation routes successfully produced CITNPs with a high catalase loading efficiency (approximately 75.5%–87.3%). CITNPs demonstrated optimal catalytic activity at 40 °C and pH 6.0–7.0. Thermal stability was markedly improved compared to free catalase, with CITNPs retaining 28.9% residual enzyme activity after exposure to 70 °C for 60 min, whereas free catalase lost approximately all activity under the same conditions. Reusability testing over 10 reaction cycles revealed sustained catalytic performance, with 41.8%–76.3% activity retained after the final cycle. In real sample applications, CITNPs effectively eliminated H2O2 artificially generated in hot tea and coffee solutions, confirming functional activity in food-associated environments. Immobilisation of catalase onto chemically modified TNPs significantly enhanced their stability and reusability without compromising enzymatic efficiency. CITNPs demonstrated strong catalytic performance even under high-temperature stress and repeated operational cycles and successfully removed H2O2 in beverage systems. These findings suggest that CITNPs are promising candidates for catalase-based applications in industrial processing, food-related systems, biotechnology, and environmental detoxification.