Enhanced Catalytic Performance and Stability of Sucrose Isomerase Via Cu²⁺-Mediated Nanoflower Immobilization

Sucrose isomerase (SIM) faces challenges in industrial applications due to its limited stability and reusability. To address these issues, a copper ion-mediated hybrid nanoflower immobilization system was developed for a rationally engineered SIM mutant (Q310E). Under optimized synthesis conditions (50 h, 120 mM Cu²⁺, SIM-to-Cu²⁺ ratio of 0.012 mg/mM, 4℃), the resulting SIM@Cu-NFs exhibited a well-defined hierarchical structure and high batch-to-batch reproducibility. The immobilized enzyme showed significantly enhanced stability, retaining over 80% activity after 1 h at pH 4.0-8.0, over 70% activity after 3 h at 50 ℃, and over 70% activity after 45 days of storage at 4℃. Moreover, it maintained 55.9% activity after 6 reuse cycles and approximately 45% after 12 cycles. Kinetic analysis revealed a 38.5% increase in catalytic efficiency (K_cat/K_m), along with improved substrate affinity (K_m decreased from 53.5 to 45.0 ± 0.7 mM) and a higher reaction rate (V_max increased from 1609 to 1950 ± 28 µM·min^- 1). These enhancements are attributed to the stabilizing effect of Cu²⁺ coordination bonds and the favorable microenvironments within the nanoflower architecture. The SIM@Cu-NF system demonstrates high operational stability, reusability, and catalytic efficiency, showing promising potential for industrial applications in enzyme-based biocatalysis.