Background: Since immobilized enzyme has to be reused several times during the process of applications, thus one has to avoid the laborious work of filtration which is also accompanied by some inevitable loss of the support by selecting magnetic nanoparticles so as to easily separated from the medium by just magnetic separation.

Results: In the present study horseradish peroxidase (HRP) was immobilized on non-modified Fe₃O₄ magnetic nanoparticles. Immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy and energy dispersive X-ray. The immobilized HRP retained 55% of its initial activity after ten reuses. The pH was shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP. The optimal temperature of soluble HRP was shifted from 40°C to 50°C for the immobilized HRP. The immobilized HRP is more thermal stable than soluble HRP. Various substrates were oxidized by immobilized HRP with higher efficiencies than those of soluble HRP. Km values of the soluble HRP and the immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H₂O₂, respectively. The effect of metals on soluble HRP and immobilized HRP has been studied. The immobilized HRP was more stable against high concentration of urea, Triton X-100 and isopropanol.

Conclusions: The physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent and water-miscible organic solvent.