# The Biocatalytic Degradation of Organic Dyes Using Laccase Immobilized Magnetic Nanoparticles
## 3 Results and Discussion  
### The Characterization of Synthesized Materials  
The immobilization conditions included (nanoparticles:enzyme ratio, glutaraldehyde concentration, and cross-linking incubation time) were optimized to get the maximum activity of the lactose enzyme. The nanoparticles:laccase ratios (\(w\)/\(w\)) were 1.0:0.5 and 1:7.0 in the case of Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\), respectively, while the concentration of glutaraldehyde as a cross-linker was 50 mM, and the incubation time was 9 h. These optimized conditions provided the maximum activities of lactose that were 90.0% and 95.0% in the case of Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\), respectively. To confirm the structure, morphology, and size of the synthesized nanomaterials, Fe\({}_{3}\)O\({}_{4}\), CuFe\({}_{2}\)O\({}_{4}\), and their nanoparticles immobilized enzyme, different characterization techniques were used, as discussed in the next section. Firstly, X-ray diffraction (XRD) was used for the characterization of synthesized free and immobilized nanoparticles, as shown in Figure 1a. According to Figure 1a, the 2-theta values with high intensities correspond to the planes (440), (511), (400), (311), and (220) for free and immobilized Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\) nanoparticles that appeared at 62.39, 57.99, 43.82, 35.25, and 33.64\({}^{\circ}\), respectively. The XRD results indicated that Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\) nanoparticles have spinel crystal structures with face-centered cubic structure, as reported in the literature [32]. The increase immobilization on the surface of the nanoparticles did not changed the phase and the nature of the synthesized nanoparticles, as indicated by the XRD results. The average size of Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\) nanoparticles, calculated using the Scherrer equation, was equal to 18.30 and 21.30 nm, respectively. Moreover, the sharp and strong XRD peaks of the nanoparticles indicated the crystallinity nature of the synthesized nanoparticles. For the study of the present functional groups in the synthesized nanoparticles, the FT-IR spectra of free Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\) nanoparticles, and their nanoparticle-immobilized increase, are shown in Figure 1b. According to Figure 1b, the Cu-O octahedral metal oxides and Fe-O tetrahedral stretching vibration bands of the synthesized CuFe\({}_{2}\)O\({}_{4}\) and Fe\({}_{3}\)O\({}_{4}\) nanoparticles were observed at 550.0 and 400.0 cm\({}^{-1}\), respectively. Free -OH stretching vibrations and CH\({}_{2}\) rocking vibrations appeared at 3421 and 813 cm\({}^{-1}\), respectively [33], in the spectra of CuFe\({}_{2}\)O\({}_{4}\) nanoparticles, Fe\({}_{3}\)O\({}_{4}\) nanoparticles, and their nanoparticle-immobilized increase. The spinel ferrite was confirmed due to the clear bands that appeared in the region of 400.0 to 600.0 cm\({}^{-1}\)[32], which confirms the XRD results. Stretching vibrations of N-H and Si-O-Fe that appeared as new bands in the spectra of nanoparticle-immobilized increase were observed at 3481 and 1201 cm\({}^{-1}\), respectively [33], and appeared due to the immobilization that followed the silanization. The attached H\({}_{2}\)O molecules on the surface of the nanoparticle-immobilized increase were represented by the band at 1651 cm\({}^{-1}\)[34], which represents the bending H-O-H vibration. Additionally, the vibrating sample magnetometer (VSM) was used to investigate the magnetic properties of the synthesized nanoparticles Fe\({}_{3}\)O\({}_{4}\) and CuFe\({}_{2}\)O\({}_{4}\), and their nanoparticle-immobilized increase, as shown in Figure 1c.