Green synthesis of multifunctional ZnFe₂O₄-MWCNT-Cellulose acetate nanocomposite for peroxidase enzyme immobilization

Herein, a multifunctional nanocomposite was developed through a green synthesis route using Dolomiaea costus extract to produce ZnFe₂O₄ nanoparticles, which were subsequently integrated with multi-walled carbon nanotubes (MWCNTs) and cellulose acetate (CA) to form a stable ZnFe₂O₄-MWCNT-CA matrix for horseradish peroxidase (HRP) immobilization. The eco-friendly synthesis avoided toxic precursors by employing plant-derived biomolecules as natural reducing and stabilizing agents. Structural and morphological analyses revealed characteristic XRD reflections of cubic spinel ZnFe₂O₄ at 2θ ≈ 30.4°, 35.4°, 54.1°, 56.6°, and 62.2°, along with graphitic carbon peaks at 24.1° and 42.8°, confirming the successful integration of ZnFe₂O₄ and MWCNTs within the cellulose acetate matrix. FTIR spectra showed metal-oxygen vibrations at 565 cm⁻¹, cellulose acetate bands at 1735 and 1040-1240 cm⁻¹, and amide bands after immobilization, while SEM-EDX confirmed a porous interconnected morphology and the presence of C, O, Fe, and Zn as the main elements. The immobilized HRP exhibited an immobilization efficiency of 82%, a 0.5-unit alkaline shift in optimum pH (7.0 to 7.5), and an enhanced optimum temperature (50 to 60 °C) compared with the free enzyme. Kinetic analysis revealed an increased apparent Km (4.6 to 10.6 mM) and Vmax (20 to 30 μmol min⁻¹), indicating reduced substrate affinity but improved catalytic turnover after immobilization. The immobilized enzyme maintained 81% activity after ten reuse cycles and retained 71% activity after 57 days of storage, outperforming comparable magnetic or carbon-based supports. These results demonstrate that the green-synthesized ZnFe₂O₄-MWCNT-CA hybrid is a robust, recyclable, and eco-compatible platform for enzyme immobilization, offering significant promise for biocatalysis, pollutant degradation, and industrial wastewater treatment applications.