The Biocatalytic Degradation of Organic Dyes Using Laccase Immobilized Magnetic Nanoparticles

: Free laccase has limitations for its use in industrial applications that require laccase immobilization on proper support, to improve its catalytic activity. Herein, the nanoparticles of magnetic iron oxide (Fe 3 O 4 ) and copper ferrite (CuFe 2 O 4 ) were successfully used as support for the immobilization of free laccase, using glutaraldehyde as a cross-linker. The immobilization conditions of laccase on the surface of nanoparticles were optimized to reach the maximum activity of the immobilized enzyme. The synthesized free nanoparticles and the nanoparticle-immobilized laccase were characterized using different techniques, including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), vibrating sample magnetometer (VSM), and thermogravimetric analysis (TGA). CuFe 2 O 4 nanoparticles, as support, enhanced laccase activity compared to free laccase and Fe 3 O 4 nanoparticle-immobilized laccase that appeared during the study of pH, temperature, and storage stability on free and immobilized laccase. The CuFe 2 O 4 and Fe 3 O 4 nanoparticle-immobilized laccase showed superior activity in a wide pH range, temperature range, and storage period, up to 20 days at 4.0 ◦ C, when compared to free laccase. Additionally, the synthesized nanobiocatalysts were examined and optimized for the biodegradation of the anionic dye Direct Red 23 (DR23). HPLC analysis was used to conﬁrm the dye degradation. The reusability of immobilized laccases for the biodegradation of DR23 dye was investigated for up to six successive cycles, with a decolorization efﬁciency over 70.0%, which indicated good reusability and excellent stability.


Introduction
The economy and development of the present world are essentially dependent on the industries. However, the other face of industrial development is the pollution of the environment, due to the release of toxic materials associated with different industries [1]. The presence of these materials is a great risk to the living ecosystem, because of their confirmed mutagenic and carcinogenic properties [2,3]. So, these harmful materials must be removed safely from different environmental systems. Dyes that are released into the environment through different printing and textile activities are an example of these dangerous pollutants [4,5]. These dyes were removed from contaminated industrial wastewater via

Synthesis of Fe 3 O 4 Magnetic Nanoparticles
The reported co-precipitation method [30] was used for the synthesis of magnetic nanoparticles as briefly discussed in this section. Firstly, we dissolved ferric chloride hexahydrate (FeCl 3 ·6H 2 O) and ferrous chloride tetrahydrate (FeCl 2 ·4H 2 O) with a molar ratio of 2:1, respectively, in 45.0 mL of deionized H 2 O with sonication. The mixture was shaken at 70.0 • C and 1000 rpm for about 35.0 min. After that, the aqueous solution of sodium hydroxide (NaOH, 25%) was added stepwise to the previous mixture until a black precipitate was formed. The black precipitate was washed three times with deionized H 2 O after being separated from the solution using an external magnet. Finally, the synthesized magnetic nanoparticles were dried at room temperature for 24 h to be ready for use.

Synthesis of Copper Ferrite (CuFe 2 O 4 ) Magnetic Nanoparticles
The co-precipitation reported method [31] was used for the synthesis of CuFe 2 O 4 nanoparticles as follows. Under an argon atmosphere, we dissolved cupric chloride dihydrate (CuCl 2 ·2H 2 O) and ferric chloride hexahydrate (FeCl 3 ·6H 2 O) in 76.0 mL deionized H 2 O with a molar ratio of 1:2, respectively, with vigorous stirring for 10.0 min. After that, an aqueous solution of sodium hydroxide (0.005 M) was added slowly to the previous mixture with continuous stirring until a black precipitate was formed. The solution was then stirred for 5 h at 90.0 • C. The black precipitate of CuFe 2 O 4 nanoparticles was washed several times with deionized H 2 O and ethyl alcohol after being separated from the reaction solution using an external magnet. Finally, the precipitate was dried for 24.0 h at 85.0 • C in the oven and calcinated for 5 h at 700.0 • C.

Immobilization of Laccase on Synthesized Nanoparticles
For the immobilization of laccase enzyme on the surface of synthesized Fe 3 O 4 and CuFe 2 O 4 nanoparticles, their surfaces were firstly amino-functionalized. This can be reached using 3-aminopropyl triethoxysilane (APTES) as a modifying agent. Then, 200.0 mL of 1:1 mixed solvent (ethanol/H 2 O) was used to disperse 1.0 g of the nanoparticles under ultra-sonication for 35.0 min. Then, the previously suspended mixtures were stirred vigorously with the gradual addition of 10.0% solution of APTES and the stirring was continued at 70.0 • C for five hours. The mixed solvent of ethanol/H 2 O was used for washing the amino-functionalized nanoparticles several times to remove any free APTES from their surfaces followed by drying the nanoparticles for 24.0 at room temperature. Then, a solution of sodium citrate buffer (pH = 4.8, 100.0 mM) was used to disperse the functionalized nanoparticles (Fe 3 O 4 or CuFe 2 O 4 ) and laccase enzyme with a nanoparticles:laccase ratio of 1.0:0.5, and 1:7.0 in the case of Fe 3 O 4 and CuFe 2 O 4 , respectively. The enzyme nanoparticles mixtures were incubated at 30.0 • C and stirred for half an hour at 150.0 rpm. After that, glutaraldehyde as a cross-linker with a concentration of 50 mM was added and incubated for 9 h with stirring at 150.0 rpm. The immobilization conditions included (nanoparticles:enzyme ratio, glutaraldehyde concentration, and cross-linking incubation time) were determined in preliminary studies to determine the optimum conditions for the immobilization. Then, the immobilized Fe 3 O 4 and CuFe 2 O 4 were washed several times with the citrate buffer after being separated with an external magnet. For the storing of the immobilized Fe 3 O 4 and CuFe 2 O 4 , the citrate buffer was used at a temperature of 4.0 • C.

Assay for Laccase Activity
ABTS as substrate was used for the determination of the activities of free laccase and nanoparticle-immobilized laccase (Fe 3 O 4 and CuFe 2 O 4 ). Sodium citrate buffer (350.0 µL, pH = 4.8, 100.0 mM) was mixed with 100.0 µL of enzyme and ABTS (50.0 µL, 10.0 mM). Then, the mixture was quietly shacked for 10.0 min at a temperature of 25.0 • C. The quantity of enzyme necessary to catalyze the oxidation of 1.0 µmol of ABTS per minute was one unit of laccase activity. UV-vis spectrophotometer (λ = 420.0 nm) was used to determine the oxidized substrate (blue-green color) as the enzyme oxidizes the ABTS to its oxidized form ABTS + . The free laccase, Fe 3 O 4 nanoparticle-immobilized laccase, and CuFe 2 O 4 nanoparticleimmobilized laccase were incubated for 60.0 min in a buffer of sodium citrate (pH = 4.8, 100.0 mM) in the temperature range of 30.0 • C to 70.0 • C to study the temperature effect. Also, the incubation of immobilized and free laccase was performed for 60.0 min at a temperature of 50.0 • C in different buffer solutions ranging from 3.0 to 9.0 to study the pH effect. The relative laccase activity after that was determined for different samples, assuming 100% for the highest activity.

Storage Stability
The free laccase, Fe 3 O 4 nanoparticle-immobilized laccase, and CuFe 2 O 4 nanoparticleimmobilized laccase were studied for storage stability by using a buffer of sodium citrate (pH = 4.8, 100.0 mM) to store the samples at a temperature of 4.0 • C for 20 days. During the 20 day storage period, the laccase activities were determined every 4 days. The laccase 100% activity was considered to be the initial activity.

Kinetic Parameters
A sodium citrate buffer (pH = 4.8, 100.0 mM) with different ABTS concentrations ranging from 0.20 mg/mL to 0.60 mg/mL was used for the calculation of free and immobilized laccase kinetic parameters, including maximum reaction rate (V max ) and Michaelis-Menten kinetic constant (K m ). Lineweaver-Burk plot was used for the calculation of kinetic parameters V max and K m from intercept and slope, respectively.

Biodegradation of DR23 Dye
The immobilized laccase was studied for the degradation of DR23 with the study of the effect of nano-biosorbents dosage in the range of 60 mg/mL to 300 mg/mL and the effect of dye concentration in the range of 10 mg/mL to 50 mg/mL. The biodegradation process was performed at a temperature of 50.0 • C and pH of 5.0 with repetition of the experiment three times to determine the errors. A UV/visible spectrophotometer was used for the determination of DR23 concentrations at λ max = 507 nm. The decolorization of the dye was calculated using the following equation: where the symbols Abs i and Abs f denote the initial absorbance and final absorbance, respectively. The reusability of the immobilized laccase on both supports (Fe 3 O 4 and CuFe 2 O 4 nanoparticles) for the degradation of DR23 was studied for up to five successive cycles. The first cycle obtained efficiency for the dye degradation that was considered to be 100.0% efficiency. The degradation conditions were adjusted as optimum conditions of pH, temperature, dosage, and dye concentration. After each cycle, the nano-biosorbent was washed several times with buffer solution after being collected from the reaction medium using an external magnet. Then, the nano-biosorbent was used to study the next cycle.

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 laccase 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 laccase 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 [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 laccase 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 laccase 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 laccase, as shown in Figure 1c. According to Figure 1c, the Fe 3 O 4 nanoparticle-immobilized laccase and CuFe 2 O 4 nanoparticle-immobilized laccase showed a reduction in the saturation magnetization (M s ) that equaled to 23.0 and 9.90 emu g −1 , respectively, when compared to the free Fe 3 O 4 nanoparticles and CuFe 2 O 4 nanoparticles, with M s values of 25.40 and 12.27 emu g −1 , respectively. The successful immobilization of nanoparticles was approved from the reduction in M s values, as the immobilized enzyme obstacles the magnetic domain of nanoparticles. There is an agreement between the magnetization results and the values in previous studies [35,36].
Also, the thermogram outline of the synthesized nanoparticles and their nanoparticleimmobilized laccase was investigated using thermogravimetric analysis (TGA), as shown in Figure 1d. In the studied temperature range (100.0 to 450.0 • C), the Fe 3 O 4 and CuFe 2 O 4 nanoparticles showed weight loss of 3.0 and 10.5%, respectively, while these values increased after the immobilization of laccase to 8.9 and 15.5%, which indicated the successful immobilization. At the temperature of 150.0 • C, an identical weight loss % was observed for the free laccase and nanoparticle-immobilized laccase, due to the evaporation of adsorbed water molecules and light molecules. When the temperature increased from 150.0 • C to 300.0 • C, the immobilized nanoparticles showed a greater weight loss %, due to the decomposition of enzymatic proteins. While at a temperature higher than 300.0 • C, the organic molecules were completely decomposed, causing the highest increase in the weight loss %. For the study of synthesized Fe 3 O 4 and CuFe 2 O 4 nanoparticles' morphologies and sizes, SEM analysis was performed, as shown in Figure 2. Figure 2a shows the SEM image of Fe 3 O 4 nanoparticles with an average particle size of 60.0 nm, while Figure 2b shows the SEM image of CuFe 2 O 4 nanoparticles with an average particle size of 50.0 nm. According to the SEM images of Fe 3 O 4 and CuFe 2 O 4 nanoparticles, the particles are sponge-like, with a spherical shape and some agglomerations. Additionally, little plate-like particles appeared in the SEM images of the nanoparticles. Figure 2c shows the SEM image of the Fe 3 O 4 nanoparticle-immobilized laccase, while Figure 2d shows the SEM image of the CuFe 2 O 4 nanoparticle-immobilized laccase. The SEM images of the nanoparticleimmobilized laccases show a large difference in morphology between the free laccase and nanoparticle-immobilized laccase, which indicated the successful immobilization process. Also, the thermogram outline of the synthesized nanoparticles and their nanoparticle-immobilized laccase was investigated using thermogravimetric analysis (TGA), as shown in Figure 1d. In the studied temperature range (100.0 to 450.0 °C ), the Fe3O4 and CuFe2O4 nanoparticles showed weight loss of 3.0 and 10.5%, respectively, while these values increased after the immobilization of laccase to 8.9 and 15.5%, which indicated the successful immobilization. At the temperature of 150.0 °C , an identical weight loss % was  particles are sponge-like, with a spherical shape and some agglomerations. Additionally little plate-like particles appeared in the SEM images of the nanoparticles. Figure 2 shows the SEM image of the Fe3O4 nanoparticle-immobilized laccase, while Figure 2d shows the SEM image of the CuFe2O4 nanoparticle-immobilized laccase. The SEM image of the nanoparticle-immobilized laccases show a large difference in morphology between the free laccase and nanoparticle-immobilized laccase, which indicated the successfu immobilization process.

The Stability of Free and Immobilized Laccase
The application of immobilized enzymes in real sample treatment requires the as sessment of the immobilized laccase's stability under severe conditions. So, the next sec tions discuss the efficiency of Fe3O4 and CuFe2O4 nanoparticles as support for the laccase enzyme, under the conditions of temperature, pH change, and storage stability.

Storage Stability of Free and Immobilized Laccase
Fe3O4 and CuFe2O4 nanoparticle-immobilized laccase, as well as free laccase, were kept for 20 days at a temperature of 4.0 °C, in a buffer solution of sodium citrate, to study their storage stability, as shown in Figure 3a. According to Figure 3a, the free laccase los

The Stability of Free and Immobilized Laccase
The application of immobilized enzymes in real sample treatment requires the assessment of the immobilized laccase's stability under severe conditions. So, the next sections discuss the efficiency of Fe 3 O 4 and CuFe 2 O 4 nanoparticles as support for the laccase enzyme, under the conditions of temperature, pH change, and storage stability.

Storage Stability of Free and Immobilized Laccase
Fe 3 O 4 and CuFe 2 O 4 nanoparticle-immobilized laccase, as well as free laccase, were kept for 20 days at a temperature of 4.0 • C, in a buffer solution of sodium citrate, to study their storage stability, as shown in Figure 3a. According to Figure 3a, the free laccase lost 71.0% of its initial activity after the storage for 20 days, while the Fe 3 O 4 and CuFe 2 O 4 nanoparticle-immobilized laccases lost only 27.0% of their initial activity within the same period. The nanoparticles of Fe 3 O 4 and CuFe 2 O 4 , as support for laccase, made the enzyme retain its activity and resist the changes in its structure. The enzymatic active sites' distortion that causes the conformational modification was prevented in the immobilized laccase, due to the contact between laccase and Fe 3 O 4 or CuFe 2 O 4 nanoparticles, by covalent linkages. Also, the CuFe 2 O 4 nanoparticle-immobilized laccase reserves its activity more than the Fe 3 O 4 nanoparticle-immobilized laccase, which indicated the great effect of copper ions on the laccase activity. This laccase stimulation occurs by the binding of copper ions to laccase enzymes via the Cu-binding sites of type-2.
laccase, due to the contact between laccase and Fe3O4 or CuFe2O4 nanoparticles, by covalent linkages. Also, the CuFe2O4 nanoparticle-immobilized laccase reserves its activity more than the Fe3O4 nanoparticle-immobilized laccase, which indicated the great effect of copper ions on the laccase activity. This laccase stimulation occurs by the binding of copper ions to laccase enzymes via the Cu-binding sites of type-2.

The pH Effect
The effect of pH on free laccase and nanoparticle-immobilized laccase was investigated in the range of 3.0 to 9.0, and the results are presented in Figure 3b. According to Figure 3b, free laccase and immobilized laccases show their maximum enzymatic activity at a pH of 5.0. So, the optimum pH of free and immobilized laccases is 5.0. Regardless of the pH of 5.0, the free laccase exhibited lower enzymatic activity than the immobilized laccases, which indicated the resistance of immobilized laccases on Fe 3 O 4 and CuFe 2 O 4 nanoparticles to the pH change compared to the free laccase. This wide pH range of immobilized laccase high activity could result from intermolecular interactions between the glutaraldehyde surrounding the active sites and the amino acid side chains of laccase. This interaction also prevents the laccase from unfolding, and subsequently resists the change in pH. Moreover, the CuFe 2 O 4 nanoparticle-immobilized laccase showed more resistance to pH change than the Fe 3 O 4 nanoparticle-immobilized laccase, due to the presence of copper ions, as discussed in the previous subsection.

The Temperature Effect
The temperature influence on free laccase and nanoparticle-immobilized laccase was investigated by the incubation for 60.0 min in a buffer solution of sodium citrate (pH = 4. nanoparticle-immobilized laccases retain 71.0% and 61.0% of their initial activity, respectively, at a temperature of 70.0 • C, while the free enzyme retains only 23.0% of its activity at the same temperature. These results indicated the resistance of laccase on nanoparticles to conformational distortion, due to the cross-linking interaction between the nanoparticles and enzymes, which makes immobilized laccase resist the changes in environmental conditions, such as high temperatures. As expected, the CuFe 2 O 4 nanoparticle-immobilized laccase showed higher resistance to temperature change than the Fe 3 O 4 nanoparticleimmobilized laccase, due to the presence of copper ions, as discussed above.

Kinetic Parameters
By varying the substrate concentrations and measuring the initial reaction rates, according to the Lineweaver and Burk plots, we calculated the kinetic parameters (maximum velocity (V max ) and Michaelis constant (K m )) for free laccase, Fe 3 O 4 immobilized laccase, and CuFe 2 O 4 nanoparticle-immobilized laccase. The kinetic parameters for the free and immobilized laccases are presented in Table 1. The affinity between the substrate and the enzyme is represented by the K m value. According to Table 1, the Fe 3 O 4 nanoparticleimmobilized laccase has the highest K m value, which is denoted by its requirement for a greater concentration of the substrate to reach the V max . The interaction between the enzymatic active sites and the substrate was hindered in the case of the immobilized enzymes, due to the steric hindrance that resulted from the nanoparticles laccase linkage and, subsequently, the K m values are higher for the immobilized laccase than the free one [37]. The irregular results of the V max and K m values in the case of the CuFe 2 O 4 nanoparticleimmobilized laccase, when compared to the free laccase, resulted from the increased activity of laccase, due to the presence of copper ions. Also, the Fe 3 O 4 nanoparticle-immobilized laccase showed the lowest value for V max , due to the reaction inhibition resulting from the accumulated product on the surfaces.

. Effect of Dye Concentration
The DR23 dye degradation using the Fe 3 O 4 and CuFe 2 O 4 nanoparticle-immobilized laccases was studied at different concentrations of the dye, ranging from 10.0 mg/mL to 50.0 mg/mL, as shown in Figure 4a. The study of dye degradation at different concentrations of the dye was performed at optimum conditions, including a pH of 5.0 and a temperature of 50.0 • C, using a nanobiocatalyst dosage of 300.0 mg/mL. According to Figure 4a, the increased dye concentration led to a decrease in the biodegradation of the dye in the case of both nanobiocatalysts. The high dye concentration may lead to failure to get the nanobiocatalyst/dye concentration to equilibrium state or, in some cases, the poisonousness of the nanobiocatalyst that causes decreased dye biodegradation with increased dye concentration [38]. Additionally, the low redox potential of the dye DR23 is responsible for the high affinity between the dye and both the nanobiocatalysts [39]. The CuFe 2 O 4 nanoparticle-immobilized laccase showed a higher extent for DR23 degradation than the Fe 3 O 4 nanoparticle-immobilized laccase, which could be attributed to the presence of copper ions. As discussed previously, the copper ions increase the catalytic properties of laccase, by binding to laccase through the copper-binding sites.
laccases was studied at different concentrations of the dye, ranging from 10.0 mg/mL to 50.0 mg/mL, as shown in Figure 4a. The study of dye degradation at different concentrations of the dye was performed at optimum conditions, including a pH of 5.0 and a temperature of 50.0 °C , using a nanobiocatalyst dosage of 300.0 mg/mL. According to Figure 4a, the increased dye concentration led to a decrease in the biodegradation of the dye in the case of both nanobiocatalysts. The high dye concentration may lead to failure to get the nanobiocatalyst/dye concentration to equilibrium state or, in some cases, the poisonousness of the nanobiocatalyst that causes decreased dye biodegradation with increased dye concentration [38]. Additionally, the low redox potential of the dye DR23 is responsible for the high affinity between the dye and both the nanobiocatalysts [39]. The CuFe2O4 nanoparticle-immobilized laccase showed a higher extent for DR23 degradation than the Fe3O4 nanoparticle-immobilized laccase, which could be attributed to the presence of copper ions. As discussed previously, the copper ions increase the catalytic properties of laccase, by binding to laccase through the copper-binding sites.

Effect of Nanobiocatalyst Dosage
DR23 dye degradation using the Fe3O4 and CuFe2O4 nanoparticle-immobilized laccases was studied at different nanobiocatalyst dosages, ranging from 60.0 mg/mL to 300.0 mg/mL, as shown in Figure 4b. The study of dye degradation at different nanobiocatalyst dosages was performed at optimum conditions, including a pH of 5.0 and a temperature of 50.0 °C , using a dye concentration of 10.0 mg/mL. According to Figure 4b

Effect of Nanobiocatalyst Dosage
DR23 dye degradation using the Fe 3 O 4 and CuFe 2 O 4 nanoparticle-immobilized laccases was studied at different nanobiocatalyst dosages, ranging from 60.0 mg/mL to 300.0 mg/mL, as shown in Figure 4b. The study of dye degradation at different nanobiocatalyst dosages was performed at optimum conditions, including a pH of 5.0 and a temperature of 50.0 • C, using a dye concentration of 10.0 mg/mL. According to Figure 4b, the increased dosage of the nanobiocatalyst caused an increase in the degradation of DR23, which could be attributed to the increased active sites of the enzymes with the added amount of the nanobiocatalyst [40]. DR23 dye showed a high affinity towards both the nanobiocatalysts. The results indicated that the most efficient dosage was 300.0 mg/L for Fe 3 O 4 nanoparticles and CuFe 2 O 4 nanoparticle-immobilized laccase, with the resultant dye degradation equal to 83.0% and 89.0%, respectively. So, the expected results were obtained, in which the CuFe 2 O 4 nanoparticles that were used as support for the laccase enzyme enhanced its catalytic activity for dye degradation, due to the presence of copper ions.

The Mechanism of DR23 Biodegradation
HPLC analysis was used to confirm the biodegradation of DR23 using CuFe 2 O 4 nanoparticle-immobilized laccase, as shown in Figure 5. The HPLC analysis of the control solution containing DR23 dye showed the peak appearance at a retention time of 0.96 min, as shown in Figure 5a. The HPLC analysis of DR23 dye using CuFe 2 O 4 nanoparticleimmobilized laccase showed a reduction in the DR23 peak that appeared at the same retention time (0.96 min), with the appearance of an additional peak at a retention time of 1.73 min, as shown in Figure 5b. The appearance of the new peak indicated the biodegradation of DR23 and the new peak was related to the degradation products. The other products of degradation may not appear in the HPLC analysis at the used wavelength [41]. The results indicated the successful biodegradation of DR23 dye using immobilized laccase on the surface of CuFe 2 O 4 nanoparticles. min, as shown in Figure 5a. The HPLC analysis of DR23 dye using CuFe2O4 nanoparticle-immobilized laccase showed a reduction in the DR23 peak that appeared at the same retention time (0.96 min), with the appearance of an additional peak at a retention time of 1.73 min, as shown in Figure 5b. The appearance of the new peak indicated the biodegradation of DR23 and the new peak was related to the degradation products. The other products of degradation may not appear in the HPLC analysis at the used wavelength [41]. The results indicated the successful biodegradation of DR23 dye using immobilized laccase on the surface of CuFe2O4 nanoparticles. The biodegradation of DR23 dye was reached through oxidation using the laccase to produce a phenolic ring that then oxidized to phenoxy radicals, and the radicals oxidized again to form carbonium ions. Then, unstable compounds were produced by the reaction of H2O with the phenolic carbon [41,42]. In the presence of oxygen, unstable compounds are removed. This biodegradation of DR23 was achieved due to the presence of copper sites in the laccase enzyme, which is responsible for the oxidation of the substrate and the reduction in oxygen to water by the type 1 site, and type 2 and 3, respectively. Hence, the The biodegradation of DR23 dye was reached through oxidation using the laccase to produce a phenolic ring that then oxidized to phenoxy radicals, and the radicals oxidized again to form carbonium ions. Then, unstable compounds were produced by the reaction of H 2 O with the phenolic carbon [41,42]. In the presence of oxygen, unstable compounds are removed. This biodegradation of DR23 was achieved due to the presence of copper sites in the laccase enzyme, which is responsible for the oxidation of the substrate and the reduction in oxygen to water by the type 1 site, and type 2 and 3, respectively. Hence, the presence of copper ions in the support enhanced the catalytic performance of the laccase enzyme. The biodegradation of DR23 produces four radicals from four substrate molecules and two H 2 O molecules from one oxygen molecule that represent the oxidation/reduction reaction [43]. The schematic representation of the azo dyes biodegradation mechanism using laccase enzyme is presented in the Supplementary Material (Scheme S1).

Reusability
Both nanoparticle-immobilized laccase (Fe 3 O 4 and CuFe 2 O 4 ) were investigated for the degradation of DR23 dye several successive times, as shown in Figure 6. The reusability has great importance in minimizing the overall cost of treatment, minimizing the enzyme wastes, and increasing its industrial applicability [44][45][46]. According to Figure 6, there is higher relative reusability of both the nanobiocatalysts towards the degradation of DR23 dye, with a gradual decrease in the efficiency of decolorization from cycle 1 to cycle 5. Cycle 1 showed the highest efficiency for both the nanobiocatalysts, while the last cycle showed the lowest efficiency.
This could attribute to the freshly used nanobiocatalyst in the first cycle. Reusing the nanobiocatalysts led to the protein leaching to the reaction medium, which caused the gradual decrease in the decolorization efficiency [47]. Also, the results indicated the higher reusability results for CuFe 2 O 4 immobilized laccase than Fe 3 O 4 immobilized laccase, indicating that CuFe 2 O 4 nanoparticles are a better support for laccase than Fe 3 O 4 nanoparticles. The decolorization efficiency of both the nanobiocatalysts was still over 70.0% after the sixth cycle, indicating the effective recovery and reusing of nanobiocatalysts. However, the CuFe 2 O 4 nanoparticles as support caused enhanced catalytic activity for the laccase enzyme. Future studies must be focused on the determination of thermodynamic parameters [48][49][50] for enzyme immobilization, to get a full image about the nanobiocatalyst.

Reusability
Both nanoparticle-immobilized laccase (Fe3O4 and CuFe2O4) were investigated for the degradation of DR23 dye several successive times, as shown in Figure 6. The reusability has great importance in minimizing the overall cost of treatment, minimizing the enzyme wastes, and increasing its industrial applicability [44][45][46]. According to Figure 6, there is higher relative reusability of both the nanobiocatalysts towards the degradation of DR23 dye, with a gradual decrease in the efficiency of decolorization from cycle 1 to cycle 5. Cycle 1 showed the highest efficiency for both the nanobiocatalysts, while the last cycle showed the lowest efficiency. Figure 6. The reusability of CuFe2O4 nanoparticle-immobilized laccase for DR23 biodegradation up to six successive cycles in optimum conditions. This could attribute to the freshly used nanobiocatalyst in the first cycle. Reusing the nanobiocatalysts led to the protein leaching to the reaction medium, which caused the gradual decrease in the decolorization efficiency [47]. Also, the results indicated the higher reusability results for CuFe2O4 immobilized laccase than Fe3O4 immobilized laccase, indicating that CuFe2O4 nanoparticles are a better support for laccase than Fe3O4 nanoparticles. The decolorization efficiency of both the nanobiocatalysts was still over 70.0% after the sixth cycle, indicating the effective recovery and reusing of nanobiocatalysts. However, the CuFe2O4 nanoparticles as support caused enhanced catalytic activity for the laccase enzyme. Future studies must be focused on the determination of thermodynamic parameters [48][49][50] for enzyme immobilization, to get a full image about the nanobiocatalyst.

Conclusions
We can conclude that the use of nanoparticles for the immobilization of the laccase enzyme through amino functionalization enhanced its catalytic activity for the successful biodegradation of DR23 dye. The CuFe 2 O 4 nanoparticle-immobilized laccase showed higher storage stability for 20 days at 4.0 • C than the free laccase and Fe 3 O 4 nanoparticleimmobilized laccase. Also, the CuFe 2 O 4 nanoparticle-immobilized laccase showed higher activity in the pH range of 3.0 to 9.0 and temperature range of 30.0 to 70.0 • C. This higher laccase activity was attributed to the presence of copper ions that stimulated the laccase activity. Additionally, the CuFe 2 O 4 nanoparticle-immobilized laccase showed higher biodegradation ability towards DR23 dye than the free laccase and Fe 3 O 4 nanoparticleimmobilized laccase. The biodegradation of DR23 dye was confirmed by the HPLC results. Also, the immobilized laccase was investigated for the biodegradation of DR23 dye for up to six successive cycles, with good reusability results. Finally, the use of nanoparticles as support for the laccase enzyme can enhance their catalytic activity and enables their applicability in industrial applications.