Green synthesis and characterization of LacTiOx nanoparticles using fungal laccase derived from coconut oil cake.

Abstract

The fungal laccase was produced by Rhizopus arrhizus using a natural liquid culture medium containing coconut oil cake as a carbon source at pH 5.5 and 32 °C for 7 days. Under optimized conditions, the enzyme exhibited a maximum laccase activity of 7.92 U/mL. The purified enzyme exhibited an apparent molecular mass of approximately 71 kDa, as determined by SDS-PAGE. The purified laccase was subsequently used for the synthesis of laccase-functionalized titanium dioxide nanoparticles (LacTiOx NPs). The synthesized nanocomposites were characterized using FTIR, SEM-EDX, TEM, XRD, XPS, DLS, TGA, DRS, and GC-MS analyses. Activity-based measurements showed an immobilization efficiency of 87.7%, an enzyme-associated protein loading of 5.25 mg/g TiO₂, and 84.2% activity recovery relative to free laccase under the assay conditions. Repeated-use experiments showed 77.9 ± 2.0% residual activity after ten consecutive catalytic cycles. The characterization results showed changes in surface morphology, hydrodynamic size, surface charge, functional groups, and thermal and optical properties following laccase association, while the anatase TiO₂ crystalline framework was retained. FTIR and XPS analyses supported the interaction of laccase-associated biomolecular components with the TiO₂ surface, although these analyses do not by themselves establish a specific molecular immobilization mechanism. DRS analysis showed a decrease in the Tauc-derived optical band-gap value from approximately 3.2 to 2.8 eV after laccase association, indicating modification of the optical absorption behaviour. GC-MS analysis tentatively identified hydrocarbons, fatty acid derivatives, amide compounds, and sulfur-containing compounds; however, their precise origin and functional contribution were not established. This study provides comprehensive physicochemical characterization of laccase-associated TiO₂ nanoparticles and demonstrates measurable enzyme association, retained catalytic activity, and repeated-use performance under the conditions investigated.

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