A robust and reusable biocatalyst for p-nitrophenol removal based on crude laccase immobilised on Fe3O4/HKUST-1

Enzyme immobilisation is a key step in translating biocatalysts into practical water treatment technologies by enhancing stability and reusability. In this study, a crude laccase extract from the white-rot fungus Trametes versicolor was immobilised via physical adsorption onto a copper-based metal–organic framework (HKUST-1) integrated with magnetic nanoparticles (Fe₃O₄/HKUST-1), yielding a stable and magnetically recoverable biocatalyst for combined adsorption and enzymatic transformation of contaminants such as p-Nitrophenol (PNP). In contrast to most reported MOF–laccase systems that rely on purified enzymes, the use of crude laccase offers a simpler and more cost-effective approach while maintaining strong catalytic performance. Physicochemical characterization (XRD, FTIR, SEM, VSM, and BET) confirmed successful immobilisation while preserving structural integrity and magnetic responsiveness. BET analysis showed decreased surface area and pore volume, consistent with partial enzyme occupation, and an immobilisation yield of 74.3% was achieved. Compared with free laccase, the immobilised system exhibited improved catalytic performance, with Km decreasing from 0.155 to 0.131 mM and Vmax increasing 2.6-fold. Under optimal conditions (pH 4.0, 50 °C), it showed 1.17-fold higher activity and enhanced thermal stability, retaining 76.7% activity after 1 h at 50 °C versus 52.5% for the free enzyme. In batch tests (20 mg/L PNP), the composite achieved 98.2% removal within 1 h and retained 67.7% efficiency after ten reuse cycles, while adsorption alone declined sharply. These results demonstrate the effectiveness of Fe₃O₄/HKUST-1 as a support for crude laccase for removing contaminants such as PNP.