Thiolation of Myco-Synthesized Fe3O4-NPs: A Novel Promising Tool for Penicillium expansium Laccase Immobilization to Decolorize Textile Dyes and as an Application for Anticancer Agent

Environmental pollution due to the continuous uncontrolled discharge of toxic dyes into the water bodies provides insight into the need to eliminate pollutants prior to discharge is significantly needed. Recently, the combination of conventional chemotherapeutic agents and nanoparticles has attracted considerable attention. Herein, the magnetic nanoparticles (Fe3O4-NPs) were synthesized using metabolites of Aspergillus niger. Further, the surfaces of Fe3O4-NPs were functionalized using 3-mercaptoproionic acid as confirmed by XRD, TEM, and SEM analyses. A purified P. expansum laccase was immobilized onto Fe3O4/3-MPA-SH and then the developed immobilized laccase (Fe3O4/3-MPA-S-S-laccase) was applied to achieve redox-mediated degradation of different dyes. The Fe3O4/3-MPA-S-S-laccase exhibited notably improved stability toward pH, temperature, organic solvents, and storage periods. The Fe3O4/3-MPA-S-S-laccase exhibited appropriate operational stability while retaining 84.34% of its initial activity after 10 cycles. The catalytic affinity (Kcat/Km) of the immobilized biocatalyst was increased above 10-fold. The experimental data showed remarkable improvement in the dyes’ decolorization using the immobilized biocatalyst in the presence of a redox mediator in seven successive cycles. Thus, the prepared novel nanocomposite-laccase can be applied as an alternative promising strategy for bioremediation of textile wastewater. The cytotoxic level of carboplatin and Fe3O4-NPs singly or in combination on various cell lines was concentration-dependent.


Introduction
Environmental pollution has been considered a daunting challenge in the twenty-first century, mainly in underdeveloped and developing countries [1][2][3]. The rapid industrialization and technological development have led to the rapid discharge of untreated toxic textile dyes and harmful contaminants into the water bodies [4,5]. The cosmetic, food, pharmaceutical, textile, printing, and leather industries are discharging recalcitrant pollutants into natural water bodies. However, water pollution produced through the discharge of untreated toxic/recalcitrant textile dyes into water bodies has severe harmful effects on the continuation of life in the specific biosphere [6,7].
Coagulation, oxidation, electrochemical precipitation, flocculation, nano-filtration, and adsorption are among the considered numerous physical and chemical technologies purchased from Sigma-Aldrich. All other reagents and chemicals used in the present research were of analytical grade. MCF-7 (human breast cancer cells), HepG2 (human hepatocellular carcinoma cells), and A549 cell lines were brought from ATCC via the holding company for biological products and vaccines (VACSERA), Cairo, Egypt.

Bio-Synthesis of Thiol-Functionalized Magnetic Nanoparticles
Magnetite (Fe 3 O 4 -NPs) nanoparticles were prepared according to [11] with definite modifications. Briefly, the A. niger (MW390925.1) used in this work was freshly inoculated into the Czapek's-Dox broth [19]. After incubation at 28 • C for 5 days, the developed pellets were excluded by filtration. Subsequently, the filtrate was then centrifuged at 10,000× g for 20 min and 4 • C. The clear supernatant was used for the biosynthesis of Fe 3 O 4 -NPs. The metallic precursor solution was attained by mixing FeSO 4 ·7H 2 O and FeCl 3 ·6H 2 O in a 1:1 ratio.
For Fe 3 O 4 -NPs biosynthesis, an aqueous metal solution (50 mL) was mixed with the same volume of fungal supernatant as the reducing and coating agent. The contents were heated at 60 • C and stirred magnetically for 2 h. The mixture pH was retained at 12 until the formation of a black color. The Fe 3 O 4 -NPs were then harvested by centrifugation for 30 min at 5000 rpm and 4 • C, washed thrice with distilled water as well as ethanol. The magnetic nanoparticles were dehydrated at 60 • C for 12 h. For functionalization, a desirable amount of magnetic nanoparticles (5 g) was mixed with 2.3 g of 3-mercaptopropanoic acid (3-MPA) by ultra-sonication in 50 mL distilled water for 12 h at ambient temperature. The preparation was kept at pH 8.0 using NaOH (0.1 M). The obtained black particles were then collected and washed with ethanol to become neutral pH. Furthermore, the black particles were pooled and dried in an oven for 20 min at 80 • C and then used for laccase immobilization [1,11,20].

Screening for the Most Potential Laccase Producer Fungal Isolate
The fungal isolates used in this work were freshly isolated from different soil samples (Benha, Egypt), and their ability to produce laccase was detected using a modified medium (MM) containing 2.0 g/L corn steep liquor; 0.07 g/L KCl; 1.2 g/L NH 4 H 2 PO 4 ; 0.5 g/L MgSO 4 ·7H 2 O; 0.1 g/L FeSO 4 ·7H 2 O supplemented with 0.5 mM (ABTS) on a rotary shaker (120 rpm) for 5 days at 28 • C. The mycelial pellets were excluded, washed, and homogenized in phosphate buffer (pH 5.0, 50 mM) for 30 min. Subsequently, the homogenate was centrifuged for 20 min at 10,000× g and 4 • C. The clear supernatant represented crude extract of laccase and the enzyme activity was measured according to [6].

Laccase Activity Assay and Protein Determination
The activity was assessed spectrophotometrically at 420 nm using 0.5 mM ABTS with the extinction coefficient (ε = 36,000 M −1 cm −1 ). The unit of laccase activity was expressed as the amount of enzyme needed for oxidizing 1 µM ABTS per min under standard assay conditions. The protein content was estimated according to the Bradford assay method [21], and compared to bovine serum albumin.

Identification and Deposition of the Most Potential Laccase-Producing Isolate
The potent isolate producing laccase used in this work was identified according to the observation of its morphological and molecular characteristics as Penicillium expansium [22]. The sequence ITS region was analyzed and then aligned with its closely related sequences at the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 5 December 2021). The FASTA sequences of the GenBank similar sequences were imported into the MEGA 10.0 portal. Then, the sequences were then aligned for the multiple sequence alignment with the ClustalW muscle algorithm. The phylogenetic tree was generated using the Neighbor-Joining method with a confidence level of 1000 bootstrap replication [6,14].

Extraction, Purification, and Molecular Homogeneity of Laccase
Extraction of laccase was performed according to [6]. Briefly, one ml (10 6 spore/mL) of P. expansum EG-MR15 (Accession number OL719228.1) was inoculated into 50 mL of MM medium supplemented with 0.5 mM ABTS, incubated at 28 • C under shaking (120 rpm) for 5 days. After incubation, the fungal pellets were excluded by filtration. The obtained filtrate was centrifuged at 10,000× g for 20 min under 4 • C. The supernatant was employed as the crude enzyme and kept at −20 • C for further work.
The laccase preparation was purified by salting out, DEAE-cellulose column (ionexchange chromatography), and Sephadex G-200 column (gel-filtration chromatography), and later used for the assay. Briefly, the crude enzyme extract was supplemented by ammonium sulfate (75% saturation) with soft stirring at 4 • C for 60 min, centrifuged at 10,000 rpm for 20 min. The precipitate was dialyzed at 4 • C for 24 h versus 20 mM phosphate buffer (pH 7.0) prepared with NaCl (150 mM). The developed concentrated laccase was applied onto a DEAE-cellulose column, after pre-equilibration with the same buffer. The active fractions were collected, dialyzed, and downstream purified using a Sephadex G-200 column. The activity of laccase was assayed according to [6]. The SDS-PAGE was used to characterize the molecular homogeneity of the purified enzyme [23]. The markers consisted of carbonic anhydrase (29 KDa), aldolase (44 KDa), ovalbumin (60 KDa), BSA (84 KDa), and acid phosphatase (100 KDa).

Immobilization of Laccase on a Thiolated Functionalized Magnetic Nanosupport
The activated nanoparticles (10 mg) were sonicated for 15 min in 20 mL of Na acetate buffer (pH 5.0, 0.1 M) before adding laccase (Lac). Subsequently, the purified laccase (1 mg/mL, 100 U/mg protein) in the same acetate buffer (10 mL, pH 5.0, 0.1 M) was added and incubated, and slowly stirred at ambient temperature for 24 h. The conjugated laccase onto thiol-activated nanosupport (Fe 3 O 4 /3-MPA-S-S-laccase) was isolated using an external magnet and washed many times using Na acetate buffer (0.1 M) until the enzyme activity disappeared from the washing solution. The laccase loading ability of the nanosupport was evaluated using different initial Lac concentrations (0.25-1.25 mg/mL). The Fe 3 O 4 /3-MPA-S-S-laccase was subjected to activity recovery percentage and biocatalyst loading capacity determination. Fe 3 O 4 /3-MPA-S-S-laccase was preserved in the same buffer at 4 • C for further use. Control beads were prepared without adding the enzymatic preparation into Fe 3 O 4 /3-MPA-SH [1].
The laccase activity was detected according to [6] with some modifications. Briefly, the reaction mixture contained 0.9 mL of Na-acetate buffer (0.1 M, pH 5.0), 1 mL of 0.5 mM ABTS, and 0.1 mL of soluble laccase (1 mg/mL), or 0.1 g of Fe 3 O 4 /3-MPA-S-S-laccase. The reaction mixtures were kept for 20 min at 28 • C. The activity recovery was calculated using the following equation [1].
where A I is the immobilized laccase activity, A Fr is the soluble laccase activity before immobilization. The level of protein bound to nanocomposite was detected by subtracting the recovered protein in the washing buffer from the protein applied for immobilization [24,25]. Laccase loading capacity was assessed by the following equation [1].
where C i is the initial protein concentration used for immobilization (mg/L), C f is the final protein concentration post immobilization (mg/L), V is the solution volume (L), and M is the nanocomposite weight (g). Furthermore, the properties of Fe 3 O 4 /3-MPA-S-S-laccase were evaluated.

Characterization of Free Laccase and Fe 3 O 4 /3-MPA-S-S-Lac pH Optima and pH Stability
The optimum pH of the free laccase and the enzyme anchored on nanocomposite was determined under standard assay conditions and using ABTS as a substrate in various pH buffers (0.1 M of sodium acetate buffer, 2.0-5.0; phosphate buffer, 6.0-7.0; Tris-HCl buffer 8.0-9.0). The highest value of laccase activity was defined as 100%. The pH stability was determined by preincubating free laccase and Fe 3 O 4 /3-MPA-S-S-laccase in respective buffers at ambient temperature for 60 min. The residual activity was assayed using ABTS in standard conditions.

Optimum Temperature and Thermal Stability
The optimum temperatures of free laccase and Fe 3 O 4 /3-MPA-S-S-laccase were investigated by incubating the reaction mixture at different temperatures (30-70 • C) using 0.5 mM ABTS in optimal pH. For the thermostability assay, the free and immobilized preparations were separately pre-incubated at the selected temperatures for 180 min. Laccase activity was determined in standard conditions at 40 • C with ABTS.

Determination of K m and V max
The maximum velocity (V max ) and Michaelis constant (K m ) values of the free and immobilized laccase were estimated by the Lineweaver-Bürk plot [26] using different concentrations (0.2-0.8 mM) of ABTS (non-phenolic substrate) and catechol (phenolic substrate) in sodium acetate buffer (pH 5.0, 0.1 M) at 40 • C.

Effect of Different Organic Solvents on Enzyme Stability
The free laccase and Fe 3 O 4 /3-MPA-S-S-laccase were incubated with various organic solvent concentrations (10-50% v/v) for 24 h at room temperature. The activity of the enzyme was subsequently determined in standard assay conditions. In parallel, the enzymatic preparations without any organic solvent were performed under the same conditions to represent the controls. The residual laccase activity was determined, relative to the corresponding control, which was defined as 100%.
Operational Stability (Reusability) The reusability of Fe 3 O 4 -NP s /3-MPA-S-S-laccase was investigated for ten consecutive cycles using 0.5 mM ABTS. Briefly, 5 mL of fresh substrate solution was mixed with 10 mg of the Fe 3 O 4 /3-MPA-S-S-laccase in 5 mL of Na acetate buffer (pH 5.0, 0.1 M) for 30 min with persistence agitation. After each cycle, the immobilized laccase was collected using a magnet and rinsed twice by Na-acetate buffer to remove the remaining ABTS and then followed by repeated trials with a fresh aliquot of the substrate. The initial activity of the Fe 3 O 4 /3-MPA-S-S-laccase was defined as 100%.

Storage Stability
The free laccase and Fe 3 O 4 /3-MPA-S-S-laccase were stored in Na acetate buffer (0.1 M, pH 5.0) at 4 • C and room temperature for 40 days. The residual activity was determined at intervals of 1,5,10,15,20,25,30, and 40 days in standard conditions. The free and immobilized laccase activities were assayed by ABTS as substrate. The residual activity of the fresh enzyme was assigned to be 100%.

Decolorization of Synthetic Dyes
The decolorization efficiency of target pollutants (synthetic dyes) from aqueous solution by free laccase, At the maximum visible λ of each dye, the concentrations of the tested dyes were chosen in order to attain absorbance around 1.0 units. In parallel, controls were performed with 15 mg of Fe 3 O 4 -NP s /3MPA-SH (without enzyme) and another with the same volume (units) of the free enzyme as the immobilized counterpart. The reaction was performed in the dark at pH 5.0, temperature 40 • C with continuous agitation to attain proper oxygenation for 6, 12, 24, 48, and 96 h. The corresponding concentration of dye before and after batch trials was monitored by a UV/visible spectrophotometer. The decolorization efficiency was defined in terms of percentage [6]. The magnetic nanocomposites were gathered by a permanent magnet after each experiment.

Reusability Assessment of Fe 3 O 4 /3-MPA-S-S-Laccase
The reusability potential of Fe 3 O 4 /3-MPA-S-S-laccase for decolorization of four toxic textile dyes was assessed for several cycles each of 24 h. After the end of each cycle, the Fe 3 O 4 -NP s /3MPA-S-S-Lac was collected by a magnet and subsequently washed with Na acetate buffer (pH 5.0). The decolorized solution was then substituted with a new dye solution to carry out the further cycles. The immobilized enzyme activity in the first cycle was assigned as 100% and the relative activity was computed for the repetitive degradation cycles.
In brief, the cells were plated before the addition of the tested compound in a sterile 96-microtiter plate and incubated for 24 h at 37 • C. The tested substances were supplemented into a growth medium containing 1 × 10 4 cells/well to attain different concentrations of carboplatin (5, 10, 15, 20 µM), Fe 3 O 4 -NPs (20, 40, 60, 80 µg/mL), and the combination of carboplatin and Fe 3 O 4 -NPs.
The test was performed in a total volume of 100 µL and the treated cells were sustained in an incubator for 24 h at 37 • C. The MTT solution (10 µL, 5 mg/mL) was introduced per well and incubated for 3 h under 5% CO 2 at 37 • C. The media were discarded and the formed purple formazan crystals were suspended using DMS (100 µL). Cells without any treatment were considered as positive control, while the medium only was negative control. Optical density after 15 min was determined at 570 nm by a microplate reader (680 XR reader, Bio-Rad, Hercules, CA, USA).

Statistical Analysis
All runs were repeated three times, and the obtained experimental data were represented as the mean value of each trial ± standard deviation (SD). Cytotoxic assay results were examined for the normality test and then one-way ANOVA was performed at a significant level of p < 0.05, then a Tukey's post-hoc test was carried out. Statistical analyses were accomplished by the Statistical Package for Social Sciences (SPSS) version 25 (IBM, Armonl, NY, USA).

Synthesis
The overall synthesis process of Fe 3 O 4 -NPs, Fe 3 O 4 /3-MPA-SH and Fe 3 O 4 /3-MPA-S-Slaccase is illustrated in Figure 1. The metallic precursors (Fe 2+ :Fe 3+ ) were firstly mixed with A. niger-fungal filtrate in order to synthesis magnetic nanocomposite which was capped with different functional groups. Further, the developed Fe 3 O 4 -NPs were surface modified with 3-MPA. It is well recognized that Fe 3 O 4 /3-MPA shows ubiquitous -SH groups on its surface. The thiolated nanocomposite (Fe 3 O 4 /3-MPA-SH) was covalently bound to the -SH groups of laccase, fabricating Fe 3 O 4 /3-MPA-S-S-Lac (immobilized laccase) through the formation of a disulfide bond. Similar results for the production of immobilized laccase through covalent bonding to thiolated supports have been described earlier [1,9]. microplate reader (680 XR reader, Bio-Rad).

Statistical Analysis
All runs were repeated three times, and the obtained experimental data were represented as the mean value of each trial ± standard deviation (SD). Cytotoxic assay results were examined for the normality test and then one-way ANOVA was performed at a significant level of p < 0.05, then a Tukey's post-hoc test was carried out. Statistical analyses were accomplished by the Statistical Package for Social Sciences (SPSS) version 25 (IBM, Armonl, NY, USA).

Synthesis
The overall synthesis process of Fe3O4-NPs, Fe3O4/3-MPA-SH and Fe3O4/3-MPA-S-Slaccase is illustrated in Figure 1. The metallic precursors (Fe 2+ : Fe 3+ ) were firstly mixed with A. niger-fungal filtrate in order to synthesis magnetic nanocomposite which was capped with different functional groups. Further, the developed Fe3O4-NPs were surface modified with 3-MPA. It is well recognized that Fe3O4/3-MPA shows ubiquitous -SH groups on its surface. The thiolated nanocomposite (Fe3O4/3-MPA-SH) was covalently bound to the -SH groups of laccase, fabricating Fe3O4/3-MPA-S-S-Lac (immobilized laccase) through the formation of a disulfide bond. Similar results for the production of immobilized laccase through covalent bonding to thiolated supports have been described earlier [1,9].

Characterization of the Nanosupport for Laccase Immobilization
Fungal biosynthesis of Fe 3 O 4 -NPs and 3-MPA capping of Fe 3 O 4 -NPs was evidently verified from FTIR ( Figure 2A). Various peaks were observed in the spectral range from 400 to 4000 cm −1 , corresponding to the plausible existence of different functional groups on the surface of the biosynthetic magnetic nanoparticles. A characteristic broad band was detected from 3420 to 3000 cm −1 which might correspond to the overlapping O-H, N-H, and aromatic hydrogen stretching vibration (Figure 2A(a). The development of inter-and intra-molecular hydrogen bonds is the possible reason for the peak shift and considerable peak width (Figure 2A(b,c)), as reported by [11]. A well-characterized peak at 1645 cm −1 was assigned to the C=N or C=O stretching vibration of amide or acid derivatives, which was shifted to a lower wavelength (Figure 2A(b,c)). A peak at 1148 cm −1 was assigned to the C-O stretching vibration. A very low-intensity peak at 1033 cm −1 could be associated with the Fe-OH vibration. The absorption peak observed at 587 cm −1 was attributed to the Fe-O-Fe stretching vibration (Figure 2A(b)). Five characteristics peaks were observed at 3416 cm −1 (overlapped N-H and O-H stretching vibration), 2687, and 2506 cm −1 (S-H stretching vibration), 1633 cm −1 (COOH), and 568 cm −1 (CSH stretching vibration), indicating the successful surface modification of the magnetic nanoparticles using 3-MPA. The presence of the carboxylic group and thiol group on the surface of the magnetic nanoparticles, confirmed the participation of fungal metabolites in the reducing and capping processes of Fe 3 O 4 -NPs and the smooth capping of 3-mercaptopropionic acid onto magnetic nanoparticles as reported by [11,29].
Upon immobilization of laccase onto Fe 3 O 4 /3-MPA-SH, two new peaks were, respectively, observed at 774 cm −1 and 624 cm −1 , hinting the S-S and C-S when compared with Fe 3 O 4 -NPs and Fe 3 O 4 /3-MPA-SH (Figure 2A(d)). During the immobilization process, it is clear that the thiolated magnetic nanoparticles reacted with the thiol group on the laccase side, forming a strong disulfide bond (-S-S-). The performance of a strong disulfide bond displayed an excellent method for immobilization. Concurring with certain research reports, the laccase was conjugated onto the thiolated chitosan composite [1,9,30]. Overall, the FTIR spectra proved the participation of the fungal extract containing biomolecules on the surface of the magnetic nanoparticles, the functionalization by 3-MPA, and are consistent with [11,20,31].
The crystalline pattern of Fe 3 O 4 -NPs and 3-MPA/Fe 3 O 4 -NP s was evaluated through the XRD analysis. Similar diffraction peaks were obtained before and after thiolation, as illustrated in Figure 2B, hinting at the crystalline nature after the smooth capping of 3mercaptopropionic acid onto Fe 3 O 4 -NPs. Six characteristic diffraction peaks ( Figure 2B Figure 2B(b)). The brooding and weak intensity principally indicates the nano-size of the magnetic particles. These findings are in agreement with those described by [12,32].
The morphological observations of the Fe 3 O 4 -NPs and Fe 3 O 4 /3-MPA-SH nanosupport were determined by SEM as shown in Figure 2C. The magnetite nanoparticles exhibited a uniform spherical structure with homogenous distribution (Figure 2C(a)). The hybrid nanocomposite (Fe 3 O 4 /3-MPA-SH) was mostly agglomerated as a result of the incorporation of 3-MPA ligands. The elemental analysis using EDX showed the intense peaks of iron, oxygen, and sulfur ( Figure 2D). Almost similar results with respect to the surface morphology and elemental distribution of the thiolated magnetite nanocomposite were detected by [1,11].
The TEM analysis of the Fe 3 O 4 -NPs and Fe 3 O 4 /3-MPA-SH nanocomposite is shown in Figure 3. The obvious spherical and quasi-polyhedral structure of Fe 3 O 4 -NPs can be observed. The 10-18 nm size of the particles was in remarkable agreement with the results detected from the XRD analysis. The granular size was increased after the performance of 3-MPA without significant fluctuations in the granular morphology ( Figure 3B). The size of Fe 3 O 4 /3-MPA-SH nanocomposite was found to be 16-20 nm. Overall, the FT-IR, SEM, EDX, and TEM analyses clearly illustrated the successful biosynthesis of Fe 3 O 4 -NPs using the fungal extract and the capping of with respect to the surface morphology and elemental distribution of the thiolated magnetite nanocomposite were detected by [1,11].  observed. The 10-18 nm size of the particles was in remarkable agreement with the results detected from the XRD analysis. The granular size was increased after the performance of 3-MPA without significant fluctuations in the granular morphology ( Figure 3B). The size of Fe3O4/3-MPA-SH nanocomposite was found to be 16-20 nm Overall, the FT-IR, SEM, EDX, and TEM analyses clearly illustrated the successfu biosynthesis of Fe3O4-NPs using the fungal extract and the capping of Fe3O4 by 3-MPA.

Screening for the Most Potent Laccase Producing Isolate
Twenty-five fungal isolates were grown on modified medium supplemented with 0.5 mM ABTS for testing their laccase productivity. The developed fungal isolates showed a plausible fluctuation in the Lac activity, of which six isolates displayed as highly active. The highest Lac activity was determined for P. expansium EG-MR15 compared to the other fungal isolates.
The morphological characterization of the most potent fungal isolate producing laccase was approved based on the sequence of the ITS region ( Figure 4). The purified amplicon was sequenced and then undergo a non-redundantly BLAST search in the NCBI database. The sequence was deposited to the NCBI database under accession number OL719228.1. The phylogenetic tree of the rDNA sequence was generated ( Figure  4B) using the Neighbor-Joining method with bootstrap replication of 1000. The isolate P expansium EG-MR15 had a 100% similarity with the isolates of P. expansium with accession number MT738591.1, MT239576.1, and MT738603.1, with zero E-value and 99% query coverage.

Screening for the Most Potent Laccase Producing Isolate
Twenty-five fungal isolates were grown on modified medium supplemented with 0.5 mM ABTS for testing their laccase productivity. The developed fungal isolates showed a plausible fluctuation in the Lac activity, of which six isolates displayed as highly active. The highest Lac activity was determined for P. expansium EG-MR15, compared to the other fungal isolates.
The morphological characterization of the most potent fungal isolate producing laccase was approved based on the sequence of the ITS region ( Figure 4). The purified amplicon was sequenced and then undergo a non-redundantly BLAST search in the NCBI database. The sequence was deposited to the NCBI database under accession number OL719228.1. The phylogenetic tree of the rDNA sequence was generated ( Figure 4B) using the Neighbor-Joining method with bootstrap replication of 1000. The isolate P. expansium EG-MR15 had a 100% similarity with the isolates of P. expansium with accession number MT738591.1, MT239576.1, and MT738603.1, with zero E-value and 99% query coverage.

Production of Purified Laccase from P. expansium
The productivity of laccase by the culture of P. expansium EG-MR15 was assessed by growing the fungal strain on a medium supplemented with 0.5 mM ABTS. Laccase was extracted and purified using ammonium sulfate (75%), DEAE-cellulose column, and Sephadex G-200 column. For each purification step, the most active fractions were pooled, concentrated, employed in the subsequent purification step, and later used for assay as mentioned previously. The specific activity of laccase by ammonium sulfate was augmented by nearly 3.18-fold associated with the crude enzyme with an overall yield of 69%. Using the DEAE-cellulose column, the specific activity of Lac was increased by nearly 14.76-fold related to the crude enzyme with a 42% overall yield. The specific activity of laccase by gel-filtration chromatography was practically improved by about 33.05-fold with an overall yield of 27%, compared with the crude enzyme. The overall purification profile of laccase from P. expansium is briefly summarized in Table 1. Hence, the purified laccase was employed for the preparation of enzyme-immobilized magnetic beads.

Production of Purified Laccase from P. expansium
The productivity of laccase by the culture of P. expansium EG-MR15 was assessed growing the fungal strain on a medium supplemented with 0.5 mM ABTS. Laccase w extracted and purified using ammonium sulfate (75%), DEAE-cellulose column, an Sephadex G-200 column. For each purification step, the most active fractions we pooled, concentrated, employed in the subsequent purification step, and later used f assay as mentioned previously. The specific activity of laccase by ammonium sulfa was augmented by nearly 3.18-fold associated with the crude enzyme with an over yield of 69%. Using the DEAE-cellulose column, the specific activity of Lac w increased by nearly 14.76-fold related to the crude enzyme with a 42% overall yield. T specific activity of laccase by gel-filtration chromatography was practically improved about 33.05-fold with an overall yield of 27%, compared with the crude enzyme. T overall purification profile of laccase from P. expansium is briefly summarized in Table  Hence, the purified laccase was employed for the preparation of enzyme-immobiliz magnetic beads.   The molecular homogeneity of purified P. expansium laccase was investigated by SDS-PAGE [14]. A single protein band of molecular mass~60 KDa was determined ( Figure 5). A previous study described Lac purification from Coriolopsis gallica with a 4.9-fold increase and an overall yield of 60.6% [33]. The molecular mass of laccase was coincident with most laccases that are monomeric proteins, as confirmed with non-denaturing PAGE, with a molecular weight of~50 to 80 KDa [6,[34][35][36].

Immobilization of Laccase on Fe3O4/3-MPA-SH
In order to authenticate the binding capacity of laccase to the thiol nanosupport, a comparative study between free laccase and immob (Fe3O4/3MPA-S-S-Lac) was conducted. The reaction was performed using preparation in Na acetate buffer (pH 5.0, 0.1 M) or 10 mg Fe3O4-3MPA acetate buffer (pH 5.0, 0.1 M). The activity of soluble laccase from P. exp ABTS was 25 U/mg protein. The results showed that the laccase immo thiolated-nanosupport, and the optimum concentration was 10 mg, w optimum with 92% activity recovery. Furthermore, the loading of lac surface of Fe3O4/3MPA-SH was assessed using various initial laccase (0.25-1.25 mg/mL).
The results in Figure 6 clearly illustrate the optimum loading capa which was determined at 1 mg/mL initial laccase concentration, hinti occupation of the Fe3O4/3-MPA-SH immobilization sites. The laccase loa (mg/g) using the above-mentioned nanosupport was superior when c other latest nanosupports used for laccase immobilization using different 2). The thiolation (-SH) of Fe3O4-NPs delivered unique binding immobilization of laccase. The -SH group of laccase is covalently boun group located over the Fe3O4/3-MPA to produce the disulfide bond as spe in turn remarkably improved the biocatalyst loading efficiency over

Immobilization of Laccase on Fe 3 O 4 /3-MPA-SH
In order to authenticate the binding capacity of laccase to the thiolated magnetic nanosupport, a comparative study between free laccase and immobilized laccase (Fe 3 O 4 / 3MPA-S-S-Lac) was conducted. The reaction was performed using 0.1 mL of free preparation in Na acetate buffer (pH 5.0, 0.1 M) or 10 mg Fe 3 O 4 -3MPA-S-S-Lac in Na acetate buffer (pH 5.0, 0.1 M). The activity of soluble laccase from P. expansum toward ABTS was 25 U/mg protein. The results showed that the laccase immobilized on the thiolated-nanosupport, and the optimum concentration was 10 mg, which was the optimum with 92% activity recovery. Furthermore, the loading of laccase onto the surface of Fe 3 O 4 /3MPA-SH was assessed using various initial laccase concentrations (0.25-1.25 mg/mL).
The results in Figure 6 clearly illustrate the optimum loading capacity of laccase, which was determined at 1 mg/mL initial laccase concentration, hinting at the best occupation of the Fe 3 O 4 /3-MPA-SH immobilization sites. The laccase loading capacity (mg/g) using the above-mentioned nanosupport was superior when compared with other latest nanosupports used for laccase immobilization using different cultures ( Table 2). The thiolation (-SH) of Fe 3 O 4 -NPs delivered unique binding sites for the immobilization of laccase. The -SH group of laccase is covalently bound to the thiol group located over the Fe 3 O 4 /3-MPA to produce the disulfide bond as speculated, which in turn remarkably improved the biocatalyst loading efficiency over the thiolated magnetic nanoparticles. Several researchers described a laccase immobilization over thiolated supports [1,9,15].

Biochemical Characterization of the Free and Immobilized Laccase
After the confirmation of the successful covalent binding of laccase over the thiolated modified magnetic nanocomposite and the biocatalyst loading capacity determination, the developed Fe3O4/3-MPA-S-S-Lac was employed for extended characterization studies.

pH Optima and pH Stability
The pH optima of Lac immobilized on the surface of thiol-functionalized Fe3O4-NPs and the free enzyme were examined by incubating the preparations in the pH range of 2-9 using various buffer systems at constant temperature. The Fe3O4/3-MPA-S-S-Lac and free laccase showed maximal activity at pH 5.0 and pH 4.0, respectively, according to Figure 7A. The pH stability of the free and immobilized biocatalyst was assayed by incubating the preparations over the range from 4.0 to 7.0 ( Figure 7B). In general, the immobilization process led to a remarkable stabilizing effect toward various pH when compared to the free one as a result of the rigidity of the conformational structure upon immobilization, related to microenvironment change [6,31].  Magnetic biochar 27 [32] Magnetized-chitosan-grafted hallohalloysite nanotube 100 [1] Magnetic-chitosan 32 [32] MACS-NIL-Cu-Laccase 47 [38] Chitosan-functionalized supermagneti cellulose 73 [39] Sepabeads EC-EP3 32.6 [40] Dilbeads NK 17.8 [40] PS: Present study.

Biochemical Characterization of the Free and Immobilized Laccase
After the confirmation of the successful covalent binding of laccase over the thiolated modified magnetic nanocomposite and the biocatalyst loading capacity determination, the developed Fe 3 O 4 /3-MPA-S-S-Lac was employed for extended characterization studies.

pH Optima and pH Stability
The pH optima of Lac immobilized on the surface of thiol-functionalized Fe 3 O 4 -NPs and the free enzyme were examined by incubating the preparations in the pH range of 2-9 using various buffer systems at constant temperature. The Fe 3 O 4 /3-MPA-S-S-Lac and free laccase showed maximal activity at pH 5.0 and pH 4.0, respectively, according to Figure 7A. The pH stability of the free and immobilized biocatalyst was assayed by incubating the preparations over the range from 4.0 to 7.0 ( Figure 7B). In general, the immobilization process led to a remarkable stabilizing effect toward various pH when compared to the free one as a result of the rigidity of the conformational structure upon immobilization, related to microenvironment change [6,31].

Temperature Optima and Thermostability
The profile of the free laccase and Fe 3 O 4 /3-MPA-S-S-Lac activity was performed by incubating the enzyme solution at various working temperatures, i.e., 30-70 • C. Laccase immobilization led to slight brooding of the activity/temperature curve, especially in the temperature range of 50 to 70 • C ( Figure 7C(a)). Thermostability is considered one of the critical challenges affecting the application of enzymes as biocatalysts in different industries. Generally, the enzyme immobilization to specific support makes it resistant to drastic conformational variations [6,12,14]. A novel support, particularly nanocomposite, has been widely applied in the enzyme immobilization processes. Thermostability of the free and immobilized laccase was performed by preincubating the biocatalyst preparation without substrate at different temperatures, i.e., 50-70 • C, at constant pH for 180 min. The solution was allowed to equilibrate for 60 min at ambient temperature and the activity was then assayed as described above. The residual activities were detected using the standard assay method.
In the present study, the immobilized laccase was more stable toward heat denaturation, with only a 5% of laccase activity loss at 50 • C when compared to the free counterpart ( Figure 7C(b)). The enhanced thermostability of immobilized enzyme could be assigned to the decline of heat transfer to enzyme microenvironment and the protection of active conformational site as a result of the covalent linking arising between laccase and the thiol functionalized magnetic nanoparticles [6,41].

Kinetic Parameters
Classical Lineweaver-Bürk plots of the free laccase and Fe 3 O 4 /3-MPA-S-S-Lac were used to evaluate the enzymatic kinetic parameters, i.e., V max , K m , and K cat by using different concentrations of ABTS (non-phenolic substrate) and catechol (phenolic substrate) as synthetic substrate under standard assay. As illustrated in Table 3, the K m values of immobilized laccase for ABTS and catechol were 2.60 and 0.93 mM, respectively, which were lower than the free enzyme (4.15 and 1.3 mM). The V max values of free and immobilized enzymes were augmented to be 29.06, 14.22 and 27.03, 14.90 Umg −1 protein for ABTS and catechol, respectively. As noticed in the Table 3, the catalytic affinity (K cat /K m ) of Fe 3 O 4 /3-MPA-S-S-Lac was increased, relative to the free enzyme. The probable reasons for kinetic parameters fluctuations are the protein rigidity, reduction in enzyme flexibility for substrate, diffusional restrictions, and slight structural changes in the substrate-binding pocket after covalent immobilization on Fe 3 O 4 /3-MPA-SH, which as a consequence enhanced the limitations of access among the enzyme and the substrate without affecting the transition state binding, hinting an increased rate of reaction [32,42,43]. Table 3. Kinetics of free and immobilized laccase. The parameters were assayed at optimal pH and temperature using different concentrations of catechol and ABTS. The diffusional coefficient (D C ) was calculated by dividing the V max value of the immobilized enzyme over the V max value of the free enzyme and used to express the magnitude of mass transfer using different substrates, namely ABTS and catechol. The diffusional coefficient was respectively found to be 0.92 and 1.05 for ABTS and catechol, which was linked to the easy accessibility (D C value more than 1.0) of the substrate into the immobilized enzyme beads [44,45].

Influence of Different Organic Solvents on Laccase Stability
For testing the stability of the free laccase and Fe 3 O 4 /3-MPA-S-S-Lac in different water-miscible solvents, the enzymatic preparations were preincubated in 1 mL of the investigated organic solutions at various concentrations (10-50%, v/v) for 24 h at ambient temperature ( Table 4). The enzyme activity was subsequently determined under standard assay conditions, relative to the enzyme preparations without any organic solvent (controls, 100%). Table 4. Stability of the free and immobilized laccase on thiolated magnetic nanoparticles in different organic solvents concentrations. In the present work, both free and immobilized enzymes exhibited a reduction in laccase activity as the concentrations of organic solvents rose. However, the Fe 3 O 4 /3-MPA-S-S-Lac showed considerably higher activity when compared with the free one at various organic solvent concentrations. Both free and immobilized laccase displayed the highest stability when using different concentrations of acetone as the organic solvent. Similar results have been reported by other investigators [46][47][48].

Operational Stability of Immobilized Laccase
The reusability of Fe 3 O 4 /3-MPA-S-S-laccase was examined because of its significant role in reducing the wastewater management processing cost. The reusability of enzymemagnetic nanocomposite in the oxidation of 0.5 mM ABTS was conducted for 10 consecutive cycles, as illustrated in Figure 8A. By the end of each cycle, the nanocomposite was easily pooled, washed, and reused for the subsequent run. The Fe 3 O 4 /3-MPA-S-S-laccase exhibited remarkable stability while retaining at 84.34% of its initial activity after 10 cycles. The activity loss in repetitive cycles of substrate oxidation might be connected to the repetitive joining of the substrate to active sites of the biocatalyst and hence, influence the binding potency between the enzyme and carrier which is linked to denaturation and inactivation of the enzyme [6,41].

Storage Stability
Generally, the enzyme preparations were not stable when evaluated over different storage periods and storage temperatures, hinting at a consequence effect on the catalytic site and a remarkable loss of its activity. The results in Figure 8B obviously illustrate that the enzyme stability was improved upon immobilization when compared with free laccase through 40 days of storage. The free and immobilized biocatalyst maintained at 4 • C retained 39.2% and 82.9% of their initial activity after 40 days of storage, respectively. Under similar storage conditions, the residual activity of both free and immobilized enzymes was respectively reduced with an approximate loss of 79.7% and 38.5% of its original activity at 28 • C. [44,48], which demonstrated that the immobilized laccases exhibited storage stability higher than the free systems when stored for the same storage time. The superior stability of the immobilized enzyme may be ascribed to the multi-point binding sites of the supports surface to the biocatalysts [32,49].

Storage Stability
Generally, the enzyme preparations were not stable when evaluated over different storage periods and storage temperatures, hinting at a consequence effect on the catalytic site and a remarkable loss of its activity. The results in Figure 8B obviously illustrate that the enzyme stability was improved upon immobilization when compared with free laccase through 40 days of storage. The free and immobilized biocatalyst maintained at 4 °C retained 39.2% and 82.9% of their initial activity after 40 days of storage, respectively. Under similar storage conditions, the residual activity of both free and immobilized enzymes was respectively reduced with an approximate loss of 79.7% and 38.5% of its original activity at 28 °C. [44,48], which demonstrated that the immobilized laccases exhibited storage stability higher than the free systems when stored for the same storage time. The superior stability of the immobilized enzyme may be ascribed to the multi-point binding sites of the supports surface to the biocatalysts [32,49].
The degradation capability of the free preparation without the redox mediator was not detected within 24 h. Meanwhile, the Fe3O4-NPs/3-MPA-SH exhibited 12.51, 15.67, 7.14, and 5.09% removal efficiency of MO, CV, RBBR, and RB-5 in the absence of the redox mediator, respectively. However, the Fe3O4/3-MPA-S-S-laccase without the redox mediator displayed no appreciable fluctuation in the removal efficiency of the target environmental pollutants, hinting at the importance of the redox mediators in the degradation systems [6] and the role of the adsorption process in the removal of dyes by enzyme-magnetized nanocomposite [1].

Biotechnological Application of Free Laccase, Fe 3 O 4 /3MPA-SH, and Fe 3 O 4 /3-MPA-S-S-Laccase for Catalytic Decolorization of Dyes
The dye decolorizing capability of the free enzyme, Fe 3 O 4 -NP s /3MPA-SH, and Fe 3 O 4 -NP s /3MPA-S-S-Lac was tested for various textile dyes from the groups of azo (MO), triarylmethane (Bb), anthraquinone (RBBR), and diazo (RB-5). The chemical structure of the target pollutants and the redox mediator are presented in Figure 9.
The degradation capability of the free preparation without the redox mediator was not detected within 24 h. Meanwhile, the Fe 3 O 4 -NP s /3-MPA-SH exhibited 12.51%, 15.67%, 7.14%, and 5.09% removal efficiency of MO, CV, RBBR, and RB-5 in the absence of the redox mediator, respectively. However, the Fe 3 O 4 /3-MPA-S-S-laccase without the redox mediator displayed no appreciable fluctuation in the removal efficiency of the target environmental pollutants, hinting at the importance of the redox mediators in the degradation systems [6] and the role of the adsorption process in the removal of dyes by enzyme-magnetized nanocomposite [1].
In the presence of the redox mediator (1-hydroxybenzotriazole), the results illustrated a remarkable decolorization capability using Fe 3 O 4 /3-MPA-S-S-laccase ( Figure 10A(D)). The decolorization level of MO by free and immobilized enzymes alone was higher than that for the other three investigated dyes in the existence of a 1 mM redox mediator. The decolorization percentage of anthraquinone and diazo by the immobilized enzyme was 30% and 14% within 24 h, respectively. Whereas the triarylmethane decolorization represented 40% within 24 h of incubation ( Figure 10A(D)). Furthermore, there was no noticeable decolorization of the anthraquinone dye RBBR and diazo dye RB-5 dye by free preparation and  In the presence of the redox mediator (1-hydroxybenzotriazole), the results illustrated a remarkable decolorization capability using Fe3O4/3-MPA-S-S-laccase (Figure mediator. The decolorization percentage of anthraquinone and diazo by the immobilized enzyme was 30% and 14% within 24 h, respectively. Whereas the triarylmethane decolorization represented 40% within 24 h of incubation ( Figure  10A(D)). Furthermore, there was no noticeable decolorization of the anthraquinone dye RBBR and diazo dye RB-5 dye by free preparation and Fe3O4-NPs/3-MPA-SH, compared with Fe3O4/3-MPA-S-S-laccase. 10A(D)). The decolorization level of MO by free and immobilized enzymes alone was higher than that for the other three investigated dyes in the existence of a 1 mM redox mediator. The decolorization percentage of anthraquinone and diazo by the immobilized enzyme was 30% and 14% within 24 h, respectively. Whereas the triarylmethane decolorization represented 40% within 24 h of incubation ( Figure  10A(D)). Furthermore, there was no noticeable decolorization of the anthraquinone dye RBBR and diazo dye RB-5 dye by free preparation and Fe3O4-NPs/3-MPA-SH, compared with Fe3O4/3-MPA-S-S-laccase. 10A(D)). The decolorization level of MO by free and immobilized enzymes alone was higher than that for the other three investigated dyes in the existence of a 1 mM redox mediator. The decolorization percentage of anthraquinone and diazo by the immobilized enzyme was 30% and 14% within 24 h, respectively. Whereas the triarylmethane decolorization represented 40% within 24 h of incubation ( Figure  10A(D)). Furthermore, there was no noticeable decolorization of the anthraquinone dye RBBR and diazo dye RB-5 dye by free preparation and Fe3O4-NPs/3-MPA-SH, compared with Fe3O4/3-MPA-S-S-laccase. The developed system (Fe3O4/3MPA-S-S-laccase) showed a remarkable decolorization potential, compared to laccase immobilized on different nanosupport systems [1,5,9]. The higher removal efficiency in the case of Fe3O4/3MPA-S-S-laccase for dyes belonging to different chromophore groups is attributed to either degradation The developed system (Fe 3 O 4 /3MPA-S-S-laccase) showed a remarkable decolorization potential, compared to laccase immobilized on different nanosupport systems [1,5,9]. The higher removal efficiency in the case of Fe 3 O 4 /3MPA-S-S-laccase for dyes belonging to different chromophore groups is attributed to either degradation using enzymes or/and biosorption of the synthetic dyes onto magnetized beads [1,6,33]. In comparison with Fe 3 O 4 /3MPA-SH and the free enzyme, the higher decolorization affinity obtained by laccase immobilized on Fe 3 O 4 -NP s /3-MPA-SH for different dyes may be linked to the activity of laccase and the availability of adsorption sites on beads with emphasis on amino, thiol, carboxylic, and hydroxyl groups, which improved the adsorption efficiency. Likewise, the steric hindrances reduced the accessibility of sulfonate, hydroxyl, and amino groups on the dye sites to laccases [44,48].
Reusability Assessment of the Covalent-Immobilized Laccase for Catalytic Decolorization of Dyes The immobilized enzymes reusability is a promising feature for wide industrial applications as it reduces the process cost [6]. In the present work, the Fe 3 O 4 /3-MPA-S-S-laccase was employed in seven successive cycles each of 24 h. After the fifth cycle, the relative decolorization rate was above 50%, except for RB-5 (41.02%) ( Figure 10E). The removal efficiency of target pollutants was gradually reduced in subsequent degradation cycles. Such a reduction in removal efficiency during additional reuse may be connected to the deactivation of the biocatalyst upon repeated batches, the outflow of biocatalyst from beads at the end of the cycle during washing, and blocking of bead pores by substrate or product [6,44,48].

Cytotoxic Effect
The HepG2, MCF-7, and A549 cells were subjected to different concentrations of carboplatin (5-20 µM) and Fe 3 O 4 -NPs (20-80 µg/mL), alone or in combination (carboplatin and Fe 3 O 4 -NPs), and the cytotoxic effect was determined using MTT analysis. Figure 11 illustrates that the toxicity level of the tested substances (carboplatin and Fe 3 O 4 -NPs) on various cell lines was concentration-dependent (p < 0.05). In the MTT assay, the combination of carboplatin and Fe 3 O 4 -NPs showed a reduction in cell viability percent. The IC 50 values of the carboplatin were 3.6, 3.4, 3.0 µM, however, they were 6.2, 4.1, 3.8 µg/mL for Fe 3 O 4 -NPs. On the other hand, the IC 50 values of the combination of carboplatin and Fe 3 O 4 -NPs were 3.3, 2.8, 2.4 µg/mL against HepG2, MCF-7, and A549, respectively. Hence, the developed nanoparticles are considered as a promising drug delivery system. These findings are in harmony with [50] who recorded that Fe 3 O 4 -NPs alone diminished the viability of PC-3 and LNCaP cells. It seems likely that the Fe 3 O 4 -NPs cytotoxicity depends on the type of cells tested which may be linked with the various redox state properties. It has been reported that Fe 3 O 4 -NPs can cause cytotoxicity through the generation of ROS which induce damage to DNA, Protein oxidation, and lipid peroxidation [51][52][53]. ROS result from the transfer of electrons to oxygen and their levels are managed by enzymatic and non-enzymatic antioxidants. The high level of ROS in cancer cells plays an important role in metastasis [54]. Excessive increases in ROS to about the threshold, resulted in cell toxicity, making some carcinogenic cells susceptible to induced apoptosis by ROS. Figure 11. Cytotoxic effect of carboplatin, Fe3O4-NPs, and combination of carboplatin and Fe3O4-NPs on the Hep G2, MCF-7, and A549 cell lines. Cell viabilities are detected using MTT assay. Cells are incubated with various concentrations of the tested compound for 24 h at 37 °C . Results are illustrated as means ± standard deviations. Different letters represent significant differences (p < 0.05) within various concentrations of the same substance. One-way ANOVA, then Tukey's HSD test was performed. n = 3 independent experiments.

Conclusions
In the present study, the Fe3O4/3-MPA-SH hybrid nanocomposite was employed as a novel nanosupport for laccase immobilization. The biogenic synthesized thiolated nanocomposite was characterized by FT-IR, XRD, SEM, and TEM analyses. Interestingly, the immobilization of laccase extracted from P. expansium on the thiolated nanosupport displayed superior stability over the soluble biocatalyst at various operating parameters (thermal, pH stability, and storage). The above-developed biocatalyst was applied to the decolorization of synthetic textile dyes in presence of a redox mediator system. The immobilized biocatalyst exhibited a significant removal rate for MO, Bb, RBBR, and RB-5. However, additional research is required to explore the efficiency of Fe3O4/3-MPA-S-S-Lac for other target pollutants under particular operating conditions. The improved recycling and regeneration of biocatalyst immobilized on the thiolated nanocomposite could be a promising advantage in the decontamination of pollutants. The above results provide insight into the performance of laccase immobilized on a novel nanosupport and suggest its use in wastewater treatment could be described as satisfactory.

Conclusions
In the present study, the Fe 3 O 4 /3-MPA-SH hybrid nanocomposite was employed as a novel nanosupport for laccase immobilization. The biogenic synthesized thiolated nanocomposite was characterized by FT-IR, XRD, SEM, and TEM analyses. Interestingly, the immobilization of laccase extracted from P. expansium on the thiolated nanosupport displayed superior stability over the soluble biocatalyst at various operating parameters (thermal, pH stability, and storage). The above-developed biocatalyst was applied to the decolorization of synthetic textile dyes in presence of a redox mediator system. The immobilized biocatalyst exhibited a significant removal rate for MO, Bb, RBBR, and RB-5. However, additional research is required to explore the efficiency of Fe 3 O 4 /3-MPA-S-S-Lac for other target pollutants under particular operating conditions. The improved recycling and regeneration of biocatalyst immobilized on the thiolated nanocomposite could be a promising advantage in the decontamination of pollutants. The above results provide insight into the performance of laccase immobilized on a novel nanosupport and suggest its use in wastewater treatment could be described as satisfactory. However, the use of nanomaterial at the industrial scale for environmental pollutant degradation has not been employed at a large scale due to their high cost and poor regeneration. Further research is needed into the use of the developed nanosupport for the immobilization of other enzymes and the application for environmental pollutants' degradation in real wastewater systems. In addition, the combination of carboplatin and Fe 3 O 4 -NPs showed a remarkable reduction in cell viability (%).