Feasibility of Combining Novel LacN with Traditional Enzyme Immobilization Techniques: Further Enhancement of Enzyme Activity and Stability

ABSTRACT: Laccase-inorganic hybrid nanoflowers (LacN) represent a novel enzyme immobilization strategy. The integration of this approach with traditional adsorption-based immobilization offers a novel and efficient pathway for laccase immobilization. In this study, LacN was immobilized onto two types of Cu/HAP-BC (natural mineral-derived Cu/nHAP-BC and porcine bone-derived Cu/pHAP-BC) to synthesize LacN@Cu/HAP-BC. Single-factor experiments, combined with Box-Behnken response surface methodology, were used to optimize the immobilization parameters. The optimal preparation conditions were determined as 60% LacN suspension, pH 6.0, reaction time of 2 h for LacN@Cu/nHAP-BC; and 80% LacN suspension, pH 6.0, reaction time of 2 h for LacN@Cu/pHAP-BC. Multiple characterizations confirmed uniform anchoring of flower-like LacN on porous Cu/HAP-BC. Compared with free laccase, LacN@Cu/HAP-BC showed significantly improved stability: after 30 d of storage, LacN@Cu/nHAP-BC and LacN@Cu/pHAP-BC retained 81.35% and 76.70% of their initial activities, respectively (vs. 42.98% for free laccase); the thermal half-lives (t_1/2) at 65 °C were 502 min and 433 min, respectively, approximately 3.0–3.5 times that of free laccase (142 min). Additionally, the two composites retained 71.98% and 80.15% of their initial activity after 10 consecutive catalytic cycles, showing satisfactory reusability. These results verify that the composites deliver favorable storage, thermal, and cyclic stability, acting as a feasible biocatalyst candidate for lab-level water treatment and biomass conversion. Further tests on continuous reactors and real wastewater pollutants are needed to confirm its industrial applicability.