Preparation and Characterization of Chitosan-Coated Manganese-Ferrite Nanoparticles Conjugated with Laccase for Environmental Bioremediation

Bioremediation with immobilized enzymes has several advantages, such as the enhancement of selectivity, activity, and stability of biocatalysts, as well as enzyme reusability. Laccase has proven to be a good candidate for the removal of a wide range of contaminants. In this study, naked or modified MnFe2O4 magnetic nanoparticles (MNPs) were used as supports for the immobilization of laccase from Trametes versicolor. To increase enzyme loading and stability, MNPs were coated with chitosan both after the MNP synthesis (MNPs-CS) and during their formation (MNPs-CSin situ). SEM analysis showed different sizes for the two coated systems, 20 nm and 10 nm for MNPs-CS and MNPs-CSin situ, respectively. After covalent immobilization of laccase by glutaraldehyde, the MNPs-CSin situ-lac and MNPs-CS-lac systems showed a good resistance to temperature denaturation and storage stability. The most promising system for use in repeated batches was MNPs-CSin situ-lac, which degraded about 80% of diclofenac compared to 70% of the free enzyme. The obtained results demonstrated that the MnFe2O4-CSin situ system could be an excellent candidate for the removal of contaminants.


Introduction
The use of enzymes in free form is generally associated with some limitations, such as high cost of production and purification, poor stability in a large range of pH and temperature, difficult recovery, and no reuse of the catalyst. Enzyme immobilization onto or within solid carriers is a way to overcome these shortcomings. Indeed, highly performant biocatalysts with better temperature and pH stability as well as with high catalytic activity, if compared to free proteins, can be obtained by using immobilization techniques [1]. The simplest method to immobilize enzymes onto surfaces is based on physical adsorption. This method takes advantage of being reversible, having low cost, and allowing the reuse of the support after enzyme inactivation. However, because physical adsorption has the inconvenience of potential enzyme release into the reaction medium, covalent binding is usually preferred. In this latter case, generally, crosslinking agents such as glutaraldehyde or epichlorohydrin are used [2].

Magnetic Nanoparticle Synthesis
Synthesis of manganese iron oxide nanoparticles (MnFe 2 O 4 ) was carried out, employing a water in toluene microemulsion procedure, as previously reported [19].
Briefly, FeN 3 O 9 ·9H 2 O and Mn(NO 3 ) 2 ·4H 2 O were dissolved in distilled water (11.5 mL) at final Fe 3+ and Mn 2+ concentrations of 0.4 M and 0.2 M, respectively. Then, a 0.4 M solution (12.5 mL) of sodium dodecylbenzenesulfate (NaDBS), previously homogenized for 15 min, was added together with excess toluene (1:20 water:toluene volume ratio). The obtained emulsion was kept for 24 h under stirring and N 2 atmosphere to avoid Mn 2+ oxidation. Finally, 20 mL of 1 M NaOH solution was added. The microemulsion, left under stirring for 2 h, was then kept at 100 • C for 90 min in a silicone oil bath (digestion process) under nitrogen flow. The precipitated powder (magnetic nanoparticles, MNPs) was collected by centrifugation and repeatedly washed with a 1:1 water:ethanol solution. After the purification step, MNPs were first dried at 40 • C and then submitted to a thermal treatment of calcination in an oven at 600 • C for 15 min.

MNP Coating with Chitosan
Magnetic nanoparticle polymer coating was carried out by two different methods: (1) CS-coating after MNP formation (CS coating); and (2) CS-coating during MNP synthesis (CS in situ). Method 1 (CS coating): 20 mg of MNPs were suspended in water (1 mL) by sonication for 10 min. Then, 1 mL of acid acetic aqueous solution (1% v/v) containing 4 mg of CS (5:1 MNPs:CS ratio) was added. After 30 min, in order to precipitate the polymer onto MNPs and restore CS amino groups, 5 mL of 1 M NaOH was added to the suspension. The coated nanoparticles were recovered by centrifugation, washed with water and THF to eliminate excess polymer, and dried in oven at 55 • C for 2 h. This nanocomposite was named MNPs-CS.
Method 2 (CS in situ): during MNP synthesis, performed as reported in Section 2.2, NaDBS was replaced with a CS acidic solution at 0.4 M concentration. MNPs were precipitated and washed as described above. The calcination process was carried out at 250 • C for 3 h instead of 600 • C for 15 min, to avoid polymer degradation. This nanocomposite was called MNPs-CS in situ .

Morphological Analysis
Morphological analysis of pristine and CS-coated MNPs was carried out by employing a high-resolution field emission scanning electron microscope (HR-FESEM, AURIGA Carl Zeiss AG, Oberkochen, Germany) combined with a Bruker X-ray Energy Dispersive Spectroscopy (EDS). This latter technique was also used to verify the Mn:Fe ratio in the MnFe 2 O 4 sample.

Laccase Immobilization onto Chitosan-Coated MNPs
Laccase immobilization onto chitosan-coated nanoparticles was performed by the support activation with glutaraldehyde. Briefly, the nanocomposite (20 mg) was put in contact with 1 mL of glutaraldehyde (1% v/v in 0.05 M phosphate buffer pH = 7.0) at 0 • C and under continuous stirring. After 3 h, MNPs were filtered and washed with distilled water in order to remove the excess of bifunctional agent. Then, a laccase solution (0.9 mL), at different concentrations, ranging from 0.20 to 1.0 mg/mL, was added to the activated nanocomposite (20 mg), and the reaction was carried out at 4 • C for 5 h, under continuous stirring. MNPs were separated from the reaction medium by a magnet. Finally, MNPs were washed with a phosphate buffer solution to remove the physically adsorbed laccase and stored at 4 • C before use.
For comparison, a physical adsorption of laccase onto MNPs, in the absence of glutaraldehyde, was also performed, following the procedure reported above and employing an enzyme concentration of 0.20 mg/mL, which resulted to be the most suitable for enzyme immobilization.

Laccase Immobilization Yield
The immobilization yield was expressed as the protein binding efficiency. The protein concentration was determined by using the modified Bradford assay [20]. Particularly, the enzyme concentrations in the solutions, initial solutions, plus those deriving from washings, were estimated from a calibration curve obtained by employing laccase as a standard protein. Briefly, 100 µL of a laccase solution containing different amounts of protein (3.7-100 µg) were added to 1 mL of the Bradford reagent and 1 mL of distilled water. The calibration curve was constructed by measuring the absorbances of the Coomassie dye-laccase complex at 620 nm, after 5 min of contact, by a UV-visible spectrophotometer.
Immobilization yield (%) was defined as: where the amount of immobilized enzyme was determined from differences between the amount of initial enzyme and the amount remaining in the solution after immobilization, plus that in washing solutions [21]. The amount of immobilized enzyme was also determined from the difference between the amount of the initial enzyme and the amount detected in washing solutions (not bonded) and normalized with respect to the initial support weight (immobilization efficiency immobilized laccase): Immobilized laccase = Amount of immobilized protein (mg) Weight of support (g)

Free and Immobilized Laccase Activity
Free and immobilized laccase activities were determined by monitoring the oxidation of 2,2 -azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) substrate by laccase, at 30 • C in 0.1 M citrate-phosphate buffer (CPB, pH = 3.0). Particularly, for the measurements of free enzyme activity, the reaction mixture contained 2 mL of an ABTS 240 µM solution, 10 µL of laccase, and CPB (690 µL) up to a total volume of 2.7 mL. For determination of immobilized laccase activity, instead, 3 mg of laccase-immobilized MNPs were put into contact with 2.7 mL of reaction mixture (2 mL of ABTS and 0.7 mL of CPB). The absorbance was measured at 420 nm, and wavelength related to the green-colored cation radical formation ABTS +· (ε = 36,000 M −1 cm −1 ) at 30 s time intervals for a total analysis time of 5 min.
The activity of laccase immobilized onto MNPs was normalized with respect to the support weight (specific activity). Particularly, the specific activity was defined as the ratio between the enzyme preparation activity (in units) and support weight: One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzed the oxidation of 1 µmoL of substrate per minute under the experimental conditions. All the experiments were carried out in triplicate.
To verify catalytic performance of laccase bonded to magnetic systems, immobilization efficiency was determined. This parameter was defined as the percentage of solid activity with respect to that deriving from the difference between protein solution activity before and after contact with MNPs (considered as immobilized activity) [21]: Additionally, the activity of the two CS-coated MNPs systems, MNPs-CS and MNPs-CS in situ , without the immobilized enzyme, was determined in order to verify the influence of the polymer coating on the nanoenzyme activity.

pH, Temperature, and Storage Stability of the Free and Immobilized Laccase
The stability of free and immobilized enzyme was studied by varying some experimental conditions. Specifically, the enzyme activity as a function of pH was determined at 30 • C, in 2.2-6.0 pH range and in 0.1 M citrate-phosphate buffer. In the same buffer, the thermal stability was instead determined by changing temperature in the 25-65 • C range. The storage stability studies were performed at 4 • C in a 0.05 M phosphate buffer (pH = 7.0). The activity assay was carried out at defined time intervals over a 30 day period.

Reusability of Immobilized Laccase
The reusability of the MNPs systems, both containing immobilized laccase and without laccase, was determined by measuring the solid activity after 5 repeated cycles. At the end of each activity assay, the sample was washed with 0.05 M phosphate buffer, pH = 7.0, and a new substrate solution was added.

Removal of a Model Drug
The potential oxidant activity of the developed systems was tested for the degradation of diclofenac (DCF), one of the most common drugs found in water as a pollutant. The degradation studies were performed at room temperature in the presence of ABTS as a mediator. The reaction mixture consisted of 10 µL DCF standard solution (5 mg/mL in methanol), 0.01 M citrate-phosphate buffer pH 3, 1 mL of 270 µM ABTS, and a proper amount of free or immobilized laccase equivalent to 0.02 U of enzymatic activity. Quantitative analysis of DCF transformation was performed by a high-performance liquid chromatographer (Shimadzu, LC-6A, Kyoto, Japan) coupled with a UV-Vis diode array detector (Shimadzu, SPD-M6A). The system was equipped with a reversed-phase column C-18 (250 × 4.6 mm 2 DI, particle size 5 µm) and a pre-column from Alltech. Injection volume and mobile phase flow were set at 20 µL and 0.6 mL/min, respectively. An isocratic Polymers 2021, 13, 1453 6 of 17 mobile phase containing H 2 O (with 1.3% of formic acid)-MeOH (40:60, v/v) was used, and the diode array detector was set at 277 nm. Additionally, in this case, the reusability of the systems with immobilized laccase was determined by measuring the solid activity after 5 repeated cycles and compared with that of both MNPs-CS systems without enzymes and free enzymes. Removal efficiency was determined as the percentage of the residual DFC which remained in solution under experimental conditions.

Nanoparticle Characterization
As reported in the Introduction, enzyme immobilization onto magnetic nanoparticles provides many advantages in biotechnological applications with respect to other types of supports. However, to stabilize the immobilized enzyme and to increase the loading capacity of the system, a functionalization of nanoparticles with small molecules or polymers is pivotal. In this study, manganese ferrite nanoparticles (MnFe 2 O 4 ), already characterized in a previous study [19], were functionalized by coating them with CS and then used for laccase immobilization. In particular, nanoparticle coating was performed both after MNP synthesis (MNPs-CS) and during their formation (MNPs-CS in situ ).

Morphological Analysis
The size and morphology of naked MnFe 2 O 4 were investigated by SEM analysis, while the CS amount present onto MNPs-CS and MNPs-CS in situ nanoparticles was determined by elemental analysis. SEM measurements evidenced a spherical morphology of the naked MNPs with 15 nm average size ( Figure 1A). MNPs size increased up to 20 nm after CS coating (image not shown), and decreased to about 10 nm when chitosan was used during nanoparticle synthesis (MNPs-CS in situ , Figure 1B). (250 × 4.6 mm 2 DI, particle size 5 μm) and a pre-column from Alltech. Injection volume and mobile phase flow were set at 20 μL and 0.6 mL/min, respectively. An isocratic mobile phase containing H2O (with 1.3% of formic acid)-MeOH (40:60, v/v) was used, and the diode array detector was set at 277 nm. Additionally, in this case, the reusability of the systems with immobilized laccase was determined by measuring the solid activity after 5 repeated cycles and compared with that of both MNPs-CS systems without enzymes and free enzymes. Removal efficiency was determined as the percentage of the residual DFC which remained in solution under experimental conditions.

Nanoparticle Characterization
As reported in the Introduction, enzyme immobilization onto magnetic nanoparticles provides many advantages in biotechnological applications with respect to other types of supports. However, to stabilize the immobilized enzyme and to increase the loading capacity of the system, a functionalization of nanoparticles with small molecules or polymers is pivotal. In this study, manganese ferrite nanoparticles (MnFe2O4), already characterized in a previous study [19], were functionalized by coating them with CS and then used for laccase immobilization. In particular, nanoparticle coating was performed both after MNP synthesis (MNPs-CS) and during their formation (MNPs-CSin situ).

Morphological Analysis
The size and morphology of naked MnFe2O4 were investigated by SEM analysis, while the CS amount present onto MNPs-CS and MNPs-CSin situ nanoparticles was determined by elemental analysis. SEM measurements evidenced a spherical morphology of the naked MNPs with 15 nm average size ( Figure 1A). MNPs size increased up to 20 nm after CS coating (image not shown), and decreased to about 10 nm when chitosan was used during nanoparticle synthesis (MNPs-CSin situ, Figure 1B).

FTIR Spectroscopy Analysis
FTIR-ATR analysis confirmed the CS coating. In Figure 2, the spectra of MnFe2O4 MNPs, chitosan, and MNPs-CS are presented. The naked nanoparticles showed characteristic peaks of M-O (metal-oxygen) stretching at 570 cm −1 , the M-OH vibration in the range 950-1050 cm −1 , a wide adsorption between 1340 and 1650 cm −1 due to both the H-O-H bending (corresponding to water incorporated in the structure) and vibrations attributed to -CH2 or -CH groups of the surfactant. Finally, a wide absorption band between 2500 and 3650 cm −1 due to the stretching of -OH was evidenced (Figure 2a) [22].

FTIR Spectroscopy Analysis
FTIR-ATR analysis confirmed the CS coating. In Figure 2, the spectra of MnFe 2 O 4 MNPs, chitosan, and MNPs-CS are presented. The naked nanoparticles showed characteristic peaks of M-O (metal-oxygen) stretching at 570 cm −1 , the M-OH vibration in the range 950-1050 cm −1 , a wide adsorption between 1340 and 1650 cm −1 due to both the H-O-H bending (corresponding to water incorporated in the structure) and vibrations attributed to -CH 2 or -CH groups of the surfactant. Finally, a wide absorption band between 2500 and 3650 cm −1 due to the stretching of -OH was evidenced (Figure 2a) [22].
In the spectrum of the MNPs-CS nanocomposite (Figure 2c), the typical abs bands of chitosan ( Figure 2b) were clearly visible. Particularly, the peaks due to th stretching were observed in the range 2800-3000 cm −1 , while the amide C=O str and the amide NH bending were present at 1650 cm −1 and 1550 cm −1 , respective sorptions in the range 1480-1200 cm −1 were attributed to the -CH2 and -CH bendi the wide peak at around 1050 cm −1 was attributed to the -C-O-C stretching [

Elemental Analysis
Elemental analysis confirmed the presence of the organic coating on MNP s ( Table 1). For the MNPs-CS sample, data evidenced a polymer content (1%) low that of the nanoparticles coated during the synthesis (4%). Probably, in this latter c smaller MNP size was responsible for the higher polymer content.

Analysis and Optimization of Laccase-Immobilized Systems
As reported by Wei and Wang, various nanomaterials can possess enzymetivity [25]. In particular, Gao et al. first reported that magnetite nanoparticles ex intrinsic peroxidase-like activity [26]. This activity is mainly related to the Fentonalytic activity of iron ions present on the surface of nanoparticles due to the inter sion of ferrous ions (Fe 2+ ) into ferric ions (Fe 3+ ) in suitable experimental conditions These nanoparticles, named nanozyme (nanomaterial-based artificial enzyme received great attention, and, to date, many studies have emerged in this field. As r by Mumtaz et al., this category includes metallic and bimetallic nanostructures In the spectrum of the MNPs-CS nanocomposite (Figure 2c), the typical absorption bands of chitosan ( Figure 2b) were clearly visible. Particularly, the peaks due to the -CH 2 stretching were observed in the range 2800-3000 cm −1 , while the amide C=O stretching and the amide NH bending were present at 1650 cm −1 and 1550 cm −1 , respectively. Absorptions in the range 1480-1200 cm −1 were attributed to the -CH 2 and -CH bending, and the wide peak at around 1050 cm −1 was attributed to the -C-O-C stretching [23,24].

Elemental Analysis
Elemental analysis confirmed the presence of the organic coating on MNP surfaces (Table 1). For the MNPs-CS sample, data evidenced a polymer content (1%) lower than that of the nanoparticles coated during the synthesis (4%). Probably, in this latter case, the smaller MNP size was responsible for the higher polymer content.

Analysis and Optimization of Laccase-Immobilized Systems
As reported by Wei and Wang, various nanomaterials can possess enzyme-like activity [25]. In particular, Gao et al. first reported that magnetite nanoparticles exhibit an intrinsic peroxidase-like activity [26]. This activity is mainly related to the Fenton-like cat- alytic activity of iron ions present on the surface of nanoparticles due to the interconversion of ferrous ions (Fe 2+ ) into ferric ions (Fe 3+ ) in suitable experimental conditions [27].
These nanoparticles, named nanozyme (nanomaterial-based artificial enzymes), have received great attention, and, to date, many studies have emerged in this field. As reported by Mumtaz et al., this category includes metallic and bimetallic nanostructures (Au, Pt, Au/Ag), metal oxide nanoparticles (CeO 2 , V 2 O 5 , CuO, MnO 2 ), metallic sulfides (FeS, Fe 3 O 4 , CuS), metal-organic frameworks (MIL-53(Fe), MIL-68), carbon-based nanomaterial (graphene oxide, carbon nanotubes), and hybrid materials (graphene oxide-Au, Pd/Fe 3 O 4 -PEI-graphene oxide) [28]. Nanozymes have been used as alternative systems for a broad range of applications thanks to their high stability and ease of functionalization [29]. Indeed, they are able to act on common substrates of many enzymes-typically, oxidoreductase and hydrolases-and for this reason, they have been widely utilized for environmental treatments, biosensing, immunoassays, and disease therapy [30]. As for the MnFe 2 O 4 nanoparticles synthesized in this work, it was found that the naked MNPs exhibited a not Fenton-type intrinsic-specific activity against ABTS of about 6.5 U/g (Figure 3), attributed to Fe 3+ ion reduction. After coating with the polysaccharide, the MNPs-CS system showed a slight increase in catalytic response (~8 U/g). These data suggested a contribution of CS to the catalytic activity of the MnFe 2 O 4 nanoparticles. Probably, owing to the polycation features, CS could promote the interaction of MNPs with the negatively charged substrate and, afterwards, ABTS could diffuse towards Fe 3+ surface ions thanks to the polymer swelling.
Au/Ag), metal oxide nanoparticles (CeO2, V2O5, CuO, MnO2), metallic sulfides (FeS, CuS), metal-organic frameworks (MIL-53(Fe), MIL-68), carbon-based nanomateria phene oxide, carbon nanotubes), and hybrid materials (graphene oxide-Au, Pd/F PEI-graphene oxide) [28]. Nanozymes have been used as alternative systems for a range of applications thanks to their high stability and ease of functionalization [2 deed, they are able to act on common substrates of many enzymes-typically, ox ductase and hydrolases-and for this reason, they have been widely utilized for en mental treatments, biosensing, immunoassays, and disease therapy [30]. As fo MnFe2O4 nanoparticles synthesized in this work, it was found that the naked MN hibited a not Fenton-type intrinsic-specific activity against ABTS of about 6.5 U/g (F 3), attributed to Fe 3+ ion reduction. After coating with the polysaccharide, the MN system showed a slight increase in catalytic response (~8 U/g). These data sugge contribution of CS to the catalytic activity of the MnFe2O4 nanoparticles. Probably, to the polycation features, CS could promote the interaction of MNPs with the nega charged substrate and, afterwards, ABTS could diffuse towards Fe 3+ surface ions t to the polymer swelling.
The MNPs-CSin situ nanoparticles, obtained by replacing NaDBS surfactant wit tosan during the synthesis, showed a specific activity of 12 U/g higher than the MN system ( Figure 3). Probably, the higher polymer amount present on the MNPs-C nanocomposite surface allowed a greater supply of the substrate and then a higher activity of the support. Thereafter, laccase was covalently bonded onto CS-coated MNPs after nanopar activation with glutaraldehyde. In the literature, various studies have investigate elucidated the role of the support on the immobilization of laccase from different so However, to the best of our knowledge, little attention has been paid to evaluatin coating influence on the loading and specific activity of Trametes versicolor laccase i bilized on magnetic nanoparticles, or on the magnetic support contribution to the ca activity of systems.
According to data in the literature, a relationship between enzymatic activity a amount of glutaraldehyde used for the activation of MNPs has been found. Particu the enzyme activity increases with increasing glutaraldehyde concentration, show maximum value at ca. 1% (v/v) concentration [31]. For this study, we chose such a co The MNPs-CS in situ nanoparticles, obtained by replacing NaDBS surfactant with chitosan during the synthesis, showed a specific activity of 12 U/g higher than the MNPs-CS system ( Figure 3). Probably, the higher polymer amount present on the MNPs-CS in situ nanocomposite surface allowed a greater supply of the substrate and then a higher redox activity of the support.
Thereafter, laccase was covalently bonded onto CS-coated MNPs after nanoparticles' activation with glutaraldehyde. In the literature, various studies have investigated and elucidated the role of the support on the immobilization of laccase from different sources. However, to the best of our knowledge, little attention has been paid to evaluating the coating influence on the loading and specific activity of Trametes versicolor laccase immobilized on magnetic nanoparticles, or on the magnetic support contribution to the catalytic activity of systems.
According to data in the literature, a relationship between enzymatic activity and the amount of glutaraldehyde used for the activation of MNPs has been found. Particularly, the enzyme activity increases with increasing glutaraldehyde concentration, showing a maximum value at ca. 1% (v/v) concentration [31]. For this study, we chose such a concentration.
To verify the influence of laccase concentration on enzyme loading, enzyme concentration was varied in the range 0.20-1 mg/mL, corresponding to 9-45 mg protein/g support. The obtained results are reported in Figure 4a. The best results were achieved at the enzyme concentration of 0.37 mg/mL (17 mg protein/g support) and 0.72 mg/mL (32 mg protein/g support). In particular, the immobilized enzyme amount was 8.3 mg of enzyme/g for MNPs-CS-lac (immobilization yield = 49%) and 2.5 mg of enzyme/g for MNPs-CS in situ -lac (immobilization yield = 8%). However, except for the 0.2 mg/mL concentration, the amount of enzyme immobilized on the support coated with CS after synthesis (MNPs-CS-lac) was higher than that bonded on the support coated during synthesis (MNPs-CS in situ -lac). This behavior could be related to the greater polymer concentration present on the MNPs-CS in situ system that probably favors nanoparticle aggregation, making binding sites for laccase less available. Moreover, generally, at higher protein concentration, the immobilization yield decreased for both developed systems, probably due to the formation of laccase aggregates, which hinder protein diffusion towards the supports. Indeed, the affinity between the cationic support and laccase, having a negative net charge, is mainly due to electrostatic interactions, which may be compromised by the formation of enzyme aggregates. In fungal laccases, at high enzyme concentration, existence of aggregate forms has been verified with a consequent reduction in active catalytic sites. Such aggregates can be associated to the enzyme "resting" form [32]. On the contrary, as for the immobilized laccase-specific activity, values around 30 U/g were found for the MNPs-CS in situ -lac system, higher than those determined for MNPs-CS-lac (Figure 4a). Overall, the specific activities found in this study are lower than those reported in the literature. Indeed, high enzyme activities were reported for laccases from Trametes versicolor and Echinodontium taxodii immobilized onto Fe 2 O 3 /SiO 2 (224U) [33] and PEG-Fe 2 O 3 (849 U/g) [34] supports, corresponding to higher amounts of protein immobilized onto solids (62.2 and 24.4 mg/g, respectively). However, the contribution of the nanoparticles alone to the catalytic activity of the systems was not studied.
In Figure 4b, immobilization efficiency vs. bonded laccase amount normalized per support weight was reported. According to obtained specific activity data, it was confirmed that the MNPs-CS in situ -lac system possessed the best performances. In addition, at low bonded enzyme amount efficiency, values higher than 100% were observed. These findings highlighted the contribution of coated support to catalytic activity ( Figure 5).
To verify if the covalent bond could affect the enzyme activity, laccase was also physically adsorbed on the supports in the absence of glutaraldehyde. Physical adsorption was performed by using an enzyme concentration of 0.2 mg/mL, because the catalytic activity of the systems with covalent laccase obtained at this binding concentration was similar to that obtained at a 0.37 mg/mL enzyme concentration. Immobilized laccase amounts of 1.5 and 1.8 mg/g were found for MNPs-CS-lac and MNPs-CS in situ -lac, respectively. The specific activity of such systems having physically adsorbed laccase was compared with that of MNPs coated with CS without enzyme or with covalently bonded enzyme, according to the two coating procedures, CS coating or CS in situ ( Figure 5).
Generally, the MNPs-CS in situ supports with or without laccase showed a specific activity higher than that of MNPs-CS. Presumably, the greater amount of CS present on the MNPs-CS in situ system, as evidenced by elemental analysis (Table 1), allowed the support to contribute significantly to the specific activity of the system, as previously explained.
In addition, the physically adsorbed laccase on both supports (MNPs-CS-lac physical) possessed a catalytic specific activity lower than that observed for the covalently immobi-lized enzyme (MNPs-CS-lac covalent). Probably, laccase was immobilized in a more active conformation when bonded to the supports.    Generally, the MNPs-CSin situ supports with or without laccase showed a specific activity higher than that of MNPs-CS. Presumably, the greater amount of CS present on the MNPs-CSin situ system, as evidenced by elemental analysis (Table 1), allowed the support to contribute significantly to the specific activity of the system, as previously explained.
In addition, the physically adsorbed laccase on both supports (MNPs-CS-lac physical) possessed a catalytic specific activity lower than that observed for the covalently immobilized enzyme (MNPs-CS-lac covalent). Probably, laccase was immobilized in a more active conformation when bonded to the supports.
The immobilization procedures of enzymes onto solid supports can provoke important structural changes that affect the stability and activity of the bonded proteins. Different spectroscopic techniques can be used to monitor structural, conformational, and electronic variations of immobilized enzymes [35]. Generally, chemical immobilization leads to a reduction in enzyme activity. However, the orientation of the protein active site on the support surface is the most important parameter influencing the activity. In our case, covalent laccase immobilization onto the coated magnetic supports probably led to an orientation of the enzyme active site more suitable for substrate bonding.

Bioreactor Characterization
The developed systems were characterized in terms of stability of pH, temperature, and time. In addition, the possibility of their reuse was evaluated. As for stability of the systems at pH variation, in agreement with the literature [36,37], the best laccase catalytic activity was found at pH = 3 both in free and immobilized forms (data not shown). As for thermal stability, generally, the enzyme immobilization on a support stabilized the biocatalyst against temperature variation. Kalkan, Nuzhet Ayca et al. observed that systems containing immobilized laccase possessed activity higher than free laccase in the range of 10-40 °C [38].
In Figure 6, the residual activity of laccase as a function of temperature ( Figure 6a) and storage time (Figure 6b) is reported. As it can be observed, the enzyme showed the maximum activity at 30 °C, both when free and immobilized. However, the stability of free laccase decreased drastically when the temperature was higher than 30 °C. In contrast, a good resistance to temperature denaturation was evidenced for the immobilized enzyme, particularly when the MNPs-CSin situ support was used for immobilization. However, in both systems, a residual activity of about 60% up to 65 °C was observed. As for The immobilization procedures of enzymes onto solid supports can provoke important structural changes that affect the stability and activity of the bonded proteins. Different spectroscopic techniques can be used to monitor structural, conformational, and electronic variations of immobilized enzymes [35]. Generally, chemical immobilization leads to a reduction in enzyme activity. However, the orientation of the protein active site on the support surface is the most important parameter influencing the activity. In our case, covalent laccase immobilization onto the coated magnetic supports probably led to an orientation of the enzyme active site more suitable for substrate bonding.

Bioreactor Characterization
The developed systems were characterized in terms of stability of pH, temperature, and time. In addition, the possibility of their reuse was evaluated. As for stability of the systems at pH variation, in agreement with the literature [36,37], the best laccase catalytic activity was found at pH = 3 both in free and immobilized forms (data not shown). As for thermal stability, generally, the enzyme immobilization on a support stabilized the biocatalyst against temperature variation. Kalkan, Nuzhet Ayca et al. observed that systems containing immobilized laccase possessed activity higher than free laccase in the range of 10-40 • C [38].
In Figure 6, the residual activity of laccase as a function of temperature ( Figure 6a) and storage time (Figure 6b) is reported. As it can be observed, the enzyme showed the maximum activity at 30 • C, both when free and immobilized. However, the stability of free laccase decreased drastically when the temperature was higher than 30 • C. In contrast, a good resistance to temperature denaturation was evidenced for the immobilized enzyme, particularly when the MNPs-CS in situ support was used for immobilization. However, in both systems, a residual activity of about 60% up to 65 • C was observed. As for time stability, free laccase lost activity over time, retaining only 28% of residual activity after 20 storage days (Figure 6b). In contrast, the enzyme immobilized onto the two CS-coated MNPs displayed remarkable stability for 30 days. These results are in accordance with data in the literature, showing that after 40 days, laccase immobilized on ferrite nanoparticles preserved a residual activity about two-fold higher (89%) than the free enzyme (49%), thus confirming the advantages of enzymatic immobilization [39].
Finally, operational stability of the developed MNPs containing covalently immobilized laccase, compared with that of MNPs without the enzyme, was evaluated in a repeated batch process (five reuse cycles, Figure 7). The MNPs-CS-lac and MNPs-CS in situ -lac systems showed a catalytic activity higher than CS-coated MNP systems without enzymes, thus evidencing the contribution of the enzyme, particularly for MNPs-CS in situ -lac. At the third cycle, the enzyme activity of MNPs-CS-lac and MNPs-CS in situ -lac was 2.5-fold and 3.8-fold higher than MNPs-CS and MNPs_CS in situ (without enzyme), respectively. However, for the MNPs-CS in situ -lac system, the residual activity result was more persistent, only showing a reduction of about 50% of the catalytic activity at the fourth cycle. Unfortunately, given the small size of the MNPs and their good dispersion in aqueous buffered medium, a loss of material after some recycling was noted. Therefore, the considerable loss of activity after the third cycle could also be attributed to the difficult recovery of the material after several reuses.
However, our results showed that the CS-coated MnFe 2 O 4 magnetic nanoparticles can be promising supports for laccase immobilization, not only for the improved thermal and storage stability, but also for the good contribution to the system's catalytic activity of the polymer coating, a very important feature in the case of enzyme denaturation or detachment. Finally, operational stability of the developed MNPs containing covalently immobilized laccase, compared with that of MNPs without the enzyme, was evaluated in a repeated batch process (five reuse cycles, Figure 7). The MNPs-CS-lac and MNPs-CSin situ-lac systems showed a catalytic activity higher than CS-coated MNP systems without en- siderable loss of activity after the third cycle could also be attributed to the difficult recovery of the material after several reuses.
However, our results showed that the CS-coated MnFe2O4 magnetic nanoparticles can be promising supports for laccase immobilization, not only for the improved thermal and storage stability, but also for the good contribution to the system's catalytic activity of the polymer coating, a very important feature in the case of enzyme denaturation or detachment.

Diclofenac Degradation
It has been widely reported that laccase, both free and immobilized, can be an efficient material for the removal of pollutants [40]. Thus, the potential applicability of the developed MNPs-CS systems containing immobilized laccase was assessed towards the degradation of diclofenac (DFC), the most common non-steroidal anti-inflammatory drug. The catalytic activity of laccase towards this class of compounds has been shown to be reduced in the absence of an appropriate mediator; therefore, the removal reaction was carried out in the presence of ABTS.
High-performance liquid chromatography (HPLC) enabled identification of the drug at every stage of its removal. Diclofenac was eluted at a retention time of 22 min ( Figure  8a). Treatment with free laccase caused an evident decrease in the well-resolved peak, with concomitant formation of a degradation product (P1) (Figure 8b).

Diclofenac Degradation
It has been widely reported that laccase, both free and immobilized, can be an efficient material for the removal of pollutants [40]. Thus, the potential applicability of the developed MNPs-CS systems containing immobilized laccase was assessed towards the degradation of diclofenac (DFC), the most common non-steroidal anti-inflammatory drug. The catalytic activity of laccase towards this class of compounds has been shown to be reduced in the absence of an appropriate mediator; therefore, the removal reaction was carried out in the presence of ABTS.
High-performance liquid chromatography (HPLC) enabled identification of the drug at every stage of its removal. Diclofenac was eluted at a retention time of 22 min (Figure 8a). Treatment with free laccase caused an evident decrease in the well-resolved peak, with concomitant formation of a degradation product (P1) (Figure 8b).
The time-course removal of diclofenac by the developed immobilized supports compared with the free enzyme is reported in Figure 9a. It was observed that all the systems possessed a catalytic action towards the drug, but with different degradation rates. The systems containing the immobilized enzyme showed a degradation higher than that of the correspondent controls (systems without laccase). In particular, the MNPs-CS-lac and MNPs-CS in situ -lac supports removed 60% and 78% of DFC, respectively, at 270 min. In addition, the MNPs-CS in situ -lac system was more efficient than the free enzyme (70% drug removal). Probably, the active conformation assumed by the enzyme after covalent immobilization together with the contribution to the catalytic activity of the polymer-coated magnetic support could justify the observed difference in DFC degradation both in terms of removal efficiency and the kinetics of degradation. The reuse of solid biocatalysts plays a key role in pollutant removal. In this case, compared with the free enzyme, the magnetic biocatalyst can be easily separated from the reaction medium by an external magnetic field and reused. The good removal efficiency of the MNPs-CS in situ -lac system was further verified by carrying out five cycles of DFC degradation (Figure 8b). The obtained findings showed that the best removal efficiency and reuse were obtained when the enzyme was immobilized on magnetic nanoparticles coated with CS during the synthesis phase. Specifically, almost no decrease in the catalytic activity was evidenced for MNPs-CS in situ -lac up to the fifth cycle. The time-course removal of diclofenac by the developed immobilized supports compared with the free enzyme is reported in Figure 9a. It was observed that all the systems possessed a catalytic action towards the drug, but with different degradation rates. The systems containing the immobilized enzyme showed a degradation higher than that of the correspondent controls (systems without laccase). In particular, the MNPs-CS-lac and MNPs-CSin situ-lac supports removed 60% and 78% of DFC, respectively, at 270 min. In addition, the MNPs-CSin situ-lac system was more efficient than the free enzyme (70% drug removal). Probably, the active conformation assumed by the enzyme after covalent immobilization together with the contribution to the catalytic activity of the polymer-coated magnetic support could justify the observed difference in DFC degradation both in terms of removal efficiency and the kinetics of degradation. The reuse of solid biocatalysts plays a key role in pollutant removal. In this case, compared with the free enzyme, the magnetic biocatalyst can be easily separated from the reaction medium by an external magnetic field and reused. The good removal efficiency of the MNPs-CSin situ-lac system was further verified by carrying out five cycles of DFC degradation (Figure 8b). The obtained findings showed that the best removal efficiency and reuse were obtained when the enzyme was immobilized on magnetic nanoparticles coated with CS during the synthesis phase. Specifically, almost no decrease in the catalytic activity was evidenced for MNPs-CSin situ-lac up to the fifth cycle.

Conclusions
Manganese iron oxide nanoparticles (MNPs) were synthesized by a reverse microemulsion method and used as supports for the covalent immobilization of laccase from Trametes versicolor. It was found that the naked MNPs exhibited a not-Fenton-type intrinsic activity against ABTS, attributed to the Fe 3+ surface ions. To avoid nanoparticle aggregation and to bind laccase on MNPs more effectively, chitosan was employed as a polymer coating and glutaraldehyde as a crosslinking agent. Polymer coating was carried out both