α-Cellulose Fibers of Paper-Waste Origin Surface-Modified with Fe3O4 and Thiolated-Chitosan for Efficacious Immobilization of Laccase

The utilization of waste-paper-biomass for extraction of important α-cellulose biopolymer, and modification of extracted α-cellulose for application in enzyme immobilization can be extremely vital for green circular bio-economy. Thus, in this study, α-cellulose fibers were super-magnetized (Fe3O4), grafted with chitosan (CTNs), and thiol (-SH) modified for laccase immobilization. The developed material was characterized by high-resolution transmission electron microscopy (HR-TEM), HR-TEM energy dispersive X-ray spectroscopy (HR-TEM-EDS), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR) analyses. Laccase immobilized on α-Cellulose-Fe3O4-CTNs (α-Cellulose-Fe3O4-CTNs-Laccase) gave significant activity recovery (99.16%) and laccase loading potential (169.36 mg/g). The α-Cellulose-Fe3O4-CTNs-Laccase displayed excellent stabilities for temperature, pH, and storage time. The α-Cellulose-Fe3O4-CTNs-Laccase applied in repeated cycles shown remarkable consistency of activity retention for 10 cycles. After the 10th cycle, α-Cellulose-Fe3O4-CTNs possessed 80.65% relative activity. Furthermore, α-Cellulose-Fe3O4-CTNs-Laccase shown excellent degradation of pharmaceutical contaminant sulfamethoxazole (SMX). The SMX degradation by α-Cellulose-Fe3O4-CTNs-Laccase was found optimum at incubation time (20 h), pH (3), temperatures (30 °C), and shaking conditions (200 rpm). Finally, α-Cellulose-Fe3O4-CTNs-Laccase gave repeated degradation of SMX. Thus, this study presents a novel, waste-derived, highly capable, and super-magnetic nanocomposite for enzyme immobilization applications.


Introduction
Paper and cardboards related waste count near about 30% of the total urban solid waste produced worldwide [1]. Despite recycling rates is higher in most of the developed countries, solid paper waste [2], and food waste [3,4], remained as a significant concern to the landfill sites. At the same time, the growing population worldwide, and the emergence of linear bio economies in addition to the growing demand for end-use products causing over-exploitation of natural resources at a rapid pace [5,6]. On average about 55% of the slurry from the paper industry globally are made from the secondary fibers called

Synthesis
The extraction of α-cellulose from paper waste biomass, the magnetization of extracted α-cellulose (α-Cellulose-Fe 3 O 4 ), and CTNs loading over magnetized α-cellulose (α-Cellulose-Fe 3 O 4 -CTNs) was carried out as previous report [33]. The thiolation of α-Cellulose-Fe 3 O 4 -CTNs was carried out in the following steps. First, α-Cellulose-Fe 3 O 4 -CTNs was washed thoroughly with distilled water to remove impurities. This well washed 100 mg of α-Cellulose-Fe 3 O 4 -CTNs was added to a 20 mL glass tube. The 15 mL of Na-acetate buffer (pH 5.0 100 mM) was added to the tube. The mixture was ultrasonicated for 15 min with power-130 W, frequency-20 kHz, and amplitude-60 µM (Sonics Vibra-Cell VC130 Ultrasonic Processor, Sonics & Materials, Inc., Newtown, CT, USA). The NHS-ester solution was prepared as per the previous report [46]. In a typical NHS-ester reaction, NHS (5.79 mM), EDAC (6.08 mM), and finally 5 mL of TGA were added to 10 mL DMF, and resulting mixture was held for shaking conditions of 200 rpm for 24 h at 25 • C. After the formation of NHS-ester, its reaction with α-Cellulose-Fe 3 O 4 -CTNs was carried out. For this, NHS-ester (0.67 mL/L) was added to ultrasonicated 15 mL solution of α-Cellulose-Fe 3 O 4 -CTNs. The mixture was kept in shaking conditions for 4 h, 25 • C, and at 200 rpm under dark conditions. After completion of this reaction, the thiolated α-Cellulose-Fe 3 O 4 -CTNs (α-Cellulose-Fe 3 O 4 -CTNs-SH) were separated magnetically. The separated α-Cellulose-Fe 3 O 4 -CTNs-SH was washed thoroughly with Na-acetate buffer (pH 5.0 100 mM) several times. The well washed α-Cellulose-Fe 3 O 4 -CTNs-SH was further applied in laccase immobilization experiments.

Laccase Immobilization on α-Cellulose-Fe 3 O 4 -CTNs-SH
The laccase (1.5 mg/mL) immobilization on α-Cellulose-Fe 3 O 4 -CTNs-SH (1 g/L) was carried out in a 20 mL glass tube containing 10 mL of Na-acetate buffer (pH 4.0, 100 mM). The mixture was gently mixed and incubated at shaking conditions of 200 rpm, 20 • C, and 24 h. After completion of the immobilization experiment, α-Cellulose-Fe3O4-CTNs-SH-Laccase was separated magnetically. The α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase was washed thoroughly with Na-acetate buffer (pH 4.0, 100 mM). The activity of laccase was done in a reaction mixture of 2 mL composed of substrate 1 mL of ABTS solution (90 µM), free laccase (0.1 mL from 1.5 mg/mL of laccase solution) or α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase (0.1 mL solution containing 1 mg of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase), and 0.9 mL of sodium acetate buffer (100 mM, pH 4). The activity testing reaction was carried out at 200 rpm, 20 • C, and for 20 min. The immobilization parameters, such as activity recovery (%), relative activity (%), and laccase loading capacity (mg/g), were measured as per equations reported in earlier reports [46]. The details of these equations are given Polymers 2021, 13, 581 4 of 17 as follows. The formulae of the measurements of activity recovery (%) were given by Equation (1): where A IL is the immobilized laccase activity, and A FL is the free laccase activity before the immobilization procedure. The laccase loading capacity on the α-Cellulose-Fe 3 O 4 -CTNs-SH (mg/g) was obtained by the Bradford method by using the Pierce™ Coomassie (Bradford) Protein Assay Kit, Thermo Scientific™, Massachusetts, MA, USA. The measurements of laccase loading capacity (mg/g) were given by Equation (2): where C bi is laccase concentration before immobilization (mg/L), C ai is retained laccase concentration in solution after immobilization (mg/L), V is the volume of the solution in liters (L), and W is the weight of α-Cellulose-Fe 3 O 4 -CTNs-SH in gram (g). Furthermore, the relative activity is given by the following Formulae (3): where A e is the activity after the stability experiment, and A i is the initial activity before the stability experiment. The effect of initial laccase concentrations 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mg/mL on activity recovery (%) and laccase loading capacity (mg/g) on α-Cellulose-Fe 3 O 4 -CTNs-SH (1 g/L) was tested in Na-acetate buffer (pH 4.0, 100 mM), at 200 rpm, 20 • C, and 24 h. Further, the thermal stability of α-Cellulose-Fe3O4-CTNs-SH-Laccase was carried out by incubating α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase and free laccase at 60 • C for 180 min in Na-acetate buffer (pH 4.0, 100 mM). The relative activity (%) was tested after each 40 min interval. The temperature for testing the temperature stability was used to be 60 • C. The reason behind this is to evaluate the stability variations in the immobilized laccase than free laccase at the higher temperature. Furthermore, the pH stability study for α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase was carried out by incubating it at various pH (1)(2)(3)(4)(5)(6)(7)(8)(9) at 200 rpm, 20 • C, and 1 h. The relative activity (%) was tested after completion of the incubation. The storage stability study was done by incubating α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase at 4 • C for 30 days. After every 5 days, samples were tested for the relative activity (%). Finally, the re-usability experiment for α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase was done. The reactants in first cycle include; substrate ABTS 1 mL (90 µM), α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase (0.1 mL solution containing 1 mg of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase), and 0.9 mL of sodium acetate buffer (100 mM, pH 4) solution. The first cycle reaction was carried out at 200 rpm, 20 • C, and for 20 min. After completion of the first cycle, resulting α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase was separated magnetically, added with a fresh reactant, and incubated at 200 rpm, 20 • C, and for 20 min to carry out the second cycle. Similar, 10 cycle reactions were carried out to assess the reusability potential of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase. The details of material characterizations were given in Supplementary Materials Method S1.

Statistical Analysis
All the values are the average of the three experiments with ± SD (standard deviation).

Statistical Analysis
All the values are the average of the three experiments with ± SD (standard deviation).

Synthesis and Strategy
The typical synthesis process and strategy of the study have been elaborated in Figure 1. The α-cellulose fibers were extracted from paper-waste, magnetized and chitosan modified (α-Cellulose-Fe3O4-CTNs) [33]. In this study, thiolation of α-Cellulose-Fe3O4-CTNs was carried out (α-Cellulose-Fe3O4-CTNs-SH) for immobilization of laccase. The typical thiolation of α-Cellulose-Fe3O4-CTNs was shown in Figure 1. The thioglycolic acid and EDAC reacts to form the unstable reactive o-acylisourea ester. The NHS reacts with the unstable reactive o-acylisourea ester to form the reactive NHS-ester. The NHS-ester attacks the amino groups of the α-Cellulose-Fe3O4-CTNs, and transfer the thiol group to form α-Cellulose-Fe3O4-CTNs-SH [46]. Further, the laccase was immobilized on the α-Cellulose-Fe3O4-CTNs-SH (α-Cellulose-Fe3O4-CTNs-SH-Laccase). This developed nano-biocatalyst α-Cellulose-Fe3O4-CTNs-SH-Laccase was further investigated the environmental application of SMX degradation ( Figure 1).  Figure 2 shows HR-TEM imaging, high-angle annular dark-field imaging (HAADF)-STEM, and EDS analysis of the α-Cellulose-Fe3O4-CTNs-SH. Figure 2A represents the surface of a particular α-cellulose loaded with Fe3O4 NPs all-round the surface area. We found  Figure 2 shows HR-TEM imaging, high-angle annular dark-field imaging (HAADF)-STEM, and EDS analysis of the α-Cellulose-Fe 3 O 4 -CTNs-SH. Figure 2A represents the surface of a particular α-cellulose loaded with Fe 3 O 4 NPs all-round the surface area. We found the Fe 3 O 4 NPs size distribution from 4 to 12 nm range with an average of about 8 nm ± 3. The shape of Fe 3 O 4 NPs were mostly observed for having circular to quasipolyhedral. The magnified HR-TEM image shown in Figure 2B confirms α-cellulose effectively provides surface area and sites for nucleation and in-situ growth of Fe 3 O 4 NPs. the Fe3O4 NPs size distribution from 4 to 12 nm range with an average of about 8 nm ± 3. The shape of Fe3O4 NPs were mostly observed for having circular to quasi-polyhedral. The magnified HR-TEM image shown in Figure 2B confirms α-cellulose effectively provides surface area and sites for nucleation and in-situ growth of Fe3O4 NPs.  The waste-paper obtained α-cellulose acted as both template and spacer to result in Fe 3 O 4 NPs having narrow distribution, without any undesirable aggregation or agglomeration. Figure Figure 2C). The elemental mapping results showed that the distribution of Fe and O in Fe 3 O 4 were consistently validated. Furthermore, it was significant to reveal that the subsequent modification of the Fe 3 O 4 loaded α-cellulose with CTNs. The presence of surface element S was clearly evident confirmation of thiolation onto the CTNs surfaces. Besides, the EDX pattern exhibited prominent peaks for iron, oxygen, carbon, and sulfur in the elemental composition of α-Cellulose-Fe 3 O 4 -CTNs-SH. This observation also confirmed the successful thiolation of α-Cellulose-Fe 3 O 4 -CTNs. As one can see in Figure 2J, α-Cellulose-Fe 3 O 4 -CTNs-SH was prepared successfully with element composition comprising, Fe (22.52%), O (18.46%), C (57.51%), N (1.05%), and S (0.04%). These all HR-TEM results confirmed that α-cellulose was successfully modified with the Fe 3 O 4 loading, CTNs grafting, and thiolation grafted chitosan.  (440), respectively as reported in the previous report [31]. The typical XRD peak for cellulose-I was observed at 14.76 attributed to the plane (110), this peak is in agreement with a previous report [49]. The characteristic XRD peaks specify that the α-cellulose surface was well decorated with Fe 3 O 4 NPs, thus clear diffraction peaks appeared with strong intensity. Thus, the XRD analysis confirms successful loading and excellent stability of Fe 3 O 4 NPs onto the α-cellulose surfaces.

XRD, VSM, XPS, and FT-IR Analysis
The magnetic properties of nano-supports developed for enzyme immobilization were highly important. As it allows to retrieve enzyme-immobilized nano-support from the reaction solution by applying external magnetic force. This removes immobilized-enzyme from the solution very easily, and it can be further applied in the next reaction or next cycle. To confirm the magnetic properties, VSM analysis of α-Cellulose-Fe 3 O 4 -CTNs-SH was performed. Figure 4 gave VSM plot of the α-Cellulose-Fe 3 O 4 -CTNs-SH. The obtained curve showed typical magnetic hysteresis with zero coercivity and remanence values. The obtained magnetic saturation value was found to be 17.38 emu/g. All these properties confirmed the super-para-magnetic nature of the α-Cellulose-Fe 3 O 4 -CTNs-SH.
XPS analysis was used to characterize the elemental profile of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase. Figure 5A shows the XPS spectra and their peaks C 1s, N 1s, O 1s, and Fe 2p, at binding energies (BEs) of 284.6, 399.70, 529.52, and 710.0 eV, respectively. Among them, peak at 399.7 eV assigns to -NH 2 of The CTNs and laccase. XPS spectra and elements confirm successful loading of the presence of Fe 3 O 4 NPs and CTNs. Furthermore, the high-resolution spectra of Fe 2p were performed for α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase by curve fitting analysis ( Figure 5B). One can see the curve fittings results obtained for Fe 2p shows two basic peaks of Fe 2p 3/2 and Fe 2p 1/2 at the BEs of 724.17 and 710.16 eV, respectively. The spin energy separation of Fe 2p 3/2 and Fe 2p 1/2 was found to be 14.01 eV, which in agreement with the previous report on Fe 3 O 4 NPs [50]. Since Fe 2p curve fitting analysis indicated the presence of both the oxidation states of Fe 2+ and Fe 3+ [51]. The XPS results revealed that Fe 3 O 4 was remained stable onto the α-cellulose ever after modified using CTNs and laccase immobilization. Polymers 2021, 13, 581 8 of 17            The surface functional groups of α-Cellulose-Fe 3 O 4 -CTNs-SH and α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase biocatalyst was examined by the FT-IR analysis, shown in Figure 6. The absorption peak observed at 442 cm −1 attributes to the Fe-O bond results from Fe 3 O 4 NPs [33]. The absorption peaks of 1116, 1318, 1450, 1631, and 3450 cm −1 obtained in all the samples, corresponds to the C-OH stretching, C-N vibrations, C-H deformation, N-H deformation, O-H stretching vibrations either from α-cellulose or CTNs as reported previously [46]. The α-cellulose materials consist of three types of atoms, carbon, oxygen, and hydrogen, which creates a straight-chain biopolymer having OH groups, and glucose rings. However, both the samples resulted in a similar peak profile consistent to the α-Cellulose-Fe 3 O 4 -CTNs-SH and α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase. However, samples after laccase enzyme immobilization resulted in FTIR peak at 1644 cm −1 (amide I) with absorption intensity owing to successful laccase enzyme immobilization. However, prominent FTIR peaks were observed for α-Cellulose-Fe 3 O 4 -CTNs-SH and α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase at 1644, 1235, and 626 cm −1 corresponds to the stretching vibrations from amides, C-SH stretching, and C-S stretching from thiol, respectively [52,53]. The appearance of the C-SH stretching peak effectively validates the thiolation of the CTNs. However, as the C-S stretching appeared in both samples, the distinctive FT-IR absorption peak for -S-S-(disulfide bond) was not observed in α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase. This might be due to the complex nature of nano-composite or overcrowding of the laccase over the surface of the α-Cellulose-Fe 3 O 4 -CTNs-SH. We found the FTIR spectral analysis in good agreement with HR-TEM, HAADF-STEM, and EDS elemental mapping results. The surface functional groups of α-Cellulose-Fe3O4-CTNs-SH and α-Cellulose-Fe3O4-CTNs-SH-Laccase biocatalyst was examined by the FT-IR analysis, shown in Figure 6. The absorption peak observed at 442 cm −1 attributes to the Fe-O bond results from Fe3O4 NPs [33]. The absorption peaks of 1116, 1318, 1450, 1631, and 3450 cm −1 obtained in all the samples, corresponds to the C-OH stretching, C-N vibrations, C-H deformation, N-H deformation, O-H stretching vibrations either from α-cellulose or CTNs as reported previously [46]. The α-cellulose materials consist of three types of atoms, carbon, oxygen, and hydrogen, which creates a straight-chain biopolymer having OH groups, and glucose rings. However, both the samples resulted in a similar peak profile consistent to the α-Cellulose-Fe3O4-CTNs-SH and α-Cellulose-Fe3O4-CTNs-SH-Laccase. However, samples after laccase enzyme immobilization resulted in FTIR peak at 1644 cm −1 (amide I) with absorption intensity owing to successful laccase enzyme immobilization. However, prominent FTIR peaks were observed for α-Cellulose-Fe3O4-CTNs-SH and α-Cellulose-Fe3O4-CTNs-SH-Laccase at 1644, 1235, and 626 cm −1 corresponds to the stretching vibrations from amides, C-SH stretching, and C-S stretching from thiol, respectively [52,53]. The appearance of the C-SH stretching peak effectively validates the thiolation of the CTNs. However, as the C-S stretching appeared in both samples, the distinctive FT-IR absorption peak for -S-S-(disulfide bond) was not observed in α-Cellulose-Fe3O4-CTNs-SH-Laccase. This might be due to the complex nature of nano-composite or overcrowding of the laccase over the surface of the α-Cellulose-Fe3O4-CTNs-SH. We found the FTIR spectral analysis in good agreement with HR-TEM, HAADF-STEM, and EDS elemental mapping results.

Activity Recovery and Laccase Loading Capacity Evaluations
The laccase loading pattern and subsequent activity recoveries with increasing initial laccase concentrations were shown in Figure 7. The laccase immobilized on α-Cellulose-Fe3O4-CTNs-SH gave laccase loading capacities of 27.87, 59.87, 83.76, 107.04, 139.89, 169.36, and 169.0 mg/g at initial laccase concentrations of 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mg/mL, respectively. Initially, with an increase in laccase concentration, the loading capacity was enhanced. At the concentration of laccase (1.8 mg/mL), loading reached a peak and remained constant further increase in the initial laccase concentration. This might be due to occupying all the immobilization sites to reach the confluence. Simultaneously, the

Activity Recovery and Laccase Loading Capacity Evaluations
The laccase loading pattern and subsequent activity recoveries with increasing initial laccase concentrations were shown in Figure 7. The laccase immobilized on α-Cellulose-Fe 3 O 4 -CTNs-SH gave laccase loading capacities of 27.87, 59.87, 83.76, 107.04, 139.89, 169.36, and 169.0 mg/g at initial laccase concentrations of 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mg/mL, respectively. Initially, with an increase in laccase concentration, the loading capacity was enhanced. At the concentration of laccase (1.8 mg/mL), loading reached a peak and remained constant further increase in the initial laccase concentration. This might be due to occupying all the immobilization sites to reach the confluence. Simultaneously, the activity recoveries of immobilized laccase was increased with an increase in initial laccase concentrations (Figure 7). The laccase activity recoveries of α-Cellulose-Fe 3 O 4 -CTNs-SH with initial laccase concentrations of 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mg/mL, were found to be 66. 22 (Figure 7). In comparison, the activity recoveries increase with an increase in the laccase loading capacity until the concentration of the 1.5 mg/mL (Figure 7). The higher enzyme activity recovery by immobilization support mainly defends upon the optimum active enzyme loading. Sometimes, a further increase in the enzyme loading causes crowding of the enzyme and results in a decrease in activity recovery. Thus, nano-support α-Cellulose-Fe 3 O 4 -CTNs-SH showed higher activity recovery with an initial laccase concentration of 1.5 mg/mL. This suggests that further increase in initial laccase concentration from the 1.5 to 1.8 mg/mL, laccase loading increases, while activity recovery remains constant. This might be due to overloading or crowding of the laccase over the surface of α-Cellulose-Fe 3 O 4 -CTNs-SH. Hence, the detailed activity recovery and laccase loading capacity data provided the information about immobilization behavior of α-Cellulose-Fe 3 O 4 -CTNs-SH. The significant laccase loading obtained by α-Cellulose-Fe 3 O 4 -CTNs-SH was quite high among the recently reported materials [33,46,[54][55][56][57][58][59][60][61]. Thus, the developed α-cellulose based material has significant potential to look like an enzyme immobilization material.
Polymers 2021, 13, 581 10 of 17 activity recoveries of immobilized laccase was increased with an increase in initial laccase concentrations (Figure 7). The laccase activity recoveries of α-Cellulose-Fe3O4-CTNs-SH with initial laccase concentrations of 0.3, 0.6, 0.9, 1.  (Figure 7). In comparison, the activity recoveries increase with an increase in the laccase loading capacity until the concentration of the 1.5 mg/mL (Figure 7). The higher enzyme activity recovery by immobilization support mainly defends upon the optimum active enzyme loading. Sometimes, a further increase in the enzyme loading causes crowding of the enzyme and results in a decrease in activity recovery. Thus, nano-support α-Cellulose-Fe3O4-CTNs-SH showed higher activity recovery with an initial laccase concentration of 1.5 mg/mL. This suggests that further increase in initial laccase concentration from the 1.5 to 1.8 mg/mL, laccase loading increases, while activity recovery remains constant. This might be due to overloading or crowding of the laccase over the surface of α-Cellulose-Fe3O4-CTNs-SH. Hence, the detailed activity recovery and laccase loading capacity data provided the information about immobilization behavior of α-Cellulose-Fe3O4-CTNs-SH. The significant laccase loading obtained by α-Cellulose-Fe3O4-CTNs-SH was quite high among the recently reported materials [33,46,[54][55][56][57][58][59][60][61]. Thus, the developed α-cellulose based material has significant potential to look like an enzyme immobilization material.
Further, the stability of free and immobilized laccase was assessed at various pH ( Figure 8B). At the incubation pH of 1, 2, 3, 4, 5, 6, 7, 8, and 9, free laccase possessed relative activities of the 6, 10, 99, 99, 88, 39, 13, 5, and 3%, and immobilized laccase shown relative activities of 80, 82, 97, 98, 91, 47, 18, 16, and 13%, respectively ( Figure 8B). These results depict enhanced pH stabilities. Particularly, at acidic pH immobilized laccase performed excellent catalysis compared to the free laccase. It is well known that the pH of the reaction is a very curtail parameter for the enzymatic reaction, hence enhancing pH stabilities at pH range (1-9) by immobilized laccase will open new facets of applications that can be exploited and studied. Thus, the α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase exhibited excellent pH stability at 1-2, and hence this immobilized system can be applied in the particular field of laccase application where acidic pH is mandatory. Similar, results of higher low pH stability obtained by previous reports [33]. The higher activity observed at the lower pH by immobilization might be due to the enhanced lower pH stability by multipoint attachment of laccase with α-Cellulose-Fe 3 O 4 -CTNs-SH. Additionally, the microenvironment of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase and bulk-solution typically takes unequal distributions of H + and OH − ion-concentrations, due to their electrostatic interactions with nano-support. This mostly results in the displacements in the pH activity profile of free and immobilized laccase.
Moreover, the storage of free enzyme solution is also one of the major concerns [38]. Long-term storage will enhance the applicability and cost-effectiveness of the biocatalyst [39]. The free and immobilized laccase was tested for storage stability by incubating it at 4 • C for 30 days. The obtained results are displayed in Figure 8C. The free laccase gave relative activity of the 78, 72, 56, 48, 44, and 37% and immobilized laccase gave 91, 83, 77, 76, 71, and 68% relative activity, at the 5, 10, 15, 20, 25, and 30 days incubation, respectively. Thus, the obtained data revealed that the α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase displayed improved storage stability over free laccase. Therefore, all the stability analysis ( Figure 8A-C) of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase corroborated better biocatalysis than free laccase. Being immobilization material developed from paper-waste material and having excellent biocatalysis performance, this will be an excellent and valuable strategy to convert waste material to the best material.

Reusability Studies of Immobilized Laccase
The industrial application of free enzymes is mostly possible in one attempt only, as the separation of products, reactant, and enzyme from solution extremely cost-effective task [38]. Hence, immobilizing the enzyme on nano-support with excellent magnetic property is significantly valuable to apply enzyme-biocatalysis in multiple cycles. Hence, in this study, laccase was immobilized on super-magnetic α-Cellulose-Fe 3 O 4 -CTNs-SH and applied in the 10 repeated cycles ( Figure 8D). The α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase gave 97, 97, 95, 90, 89, 88, 87, 86, 83, and 80% relative activity. The obtained reusability results are highly encouraging and important to apply laccase biocatalysis for various applications. Thus, all these biocatalysis studies confirmed the potential of the α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase as a novel, cost-effective, and environmental-friendly biocatalyst for laccase based applications.

Degradation of SMX by α-Cellulose-Fe3O4-CTNs-SH-Laccase
Laccase is a well-known biocatalyst for the degradation of environmental pollutants [60,61]. The pharmaceutical compound "SMX" causes serious environmental and health issues [61]. Hence, in this study immobilized laccase was tested for the degradation of SMX. The degradation of SMX by α-Cellulose-Fe3O4-CTNs-SH-Laccase was assessed for incubation time 4 to 24 h ( Figure 9A

Degradation of SMX by α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase
Laccase is a well-known biocatalyst for the degradation of environmental pollutants [60,61]. The pharmaceutical compound "SMX" causes serious environmental and health issues [61]. Hence, in this study immobilized laccase was tested for the degradation of SMX. of 2-4. After understanding the effect of pH, the effect of temperature was assessed for α-Cellulose-Fe3O4-CTNs-SH-Laccase mediated degradation of the SMX (Figure 9C). At the temperatures of 20, 30, 40, and 50, SMX degradation of 81, 83, 61, and 50 was observed. The higher SMX degradation was observed at 30 °C. Moreover, the effect of shaking conditions on the SMX degradation by α-Cellulose-Fe3O4-CTNs-SH-Laccase was displayed in ( Figure 9D). The shaking conditions of 50, 100, 150, 200, and 250 rpm yielded 79, 80, 81, 83, and 79% SMX degradation. The higher degradation was observed at 200 rpm. However, not significant difference or effect of shaking condition was observed on the degradation of SMX by α-Cellulose-Fe3O4-CTNs-SH-Laccase. Thus, all the optimized parameters; incubation time, pH, temperatures, and shaking conditions were highly important to scale-up of the treatment protocol for α-Cellulose-Fe3O4-CTNs-SH-Laccase mediated SMX degradation. Furthermore, the repeated degradation of SMX by α-Cellulose-Fe3O4-CTNs-SH-Laccase was performed in a repeated cycle experiment ( Figure 10). The free laccase can access its application of SMX degradation in only one cycle. After completion of the first degradation cycle, α-Cellulose-Fe3O4-CTNs-SH-Laccase was retrieved by an external magnet, washed thoroughly with acetate buffer solution (100 mM, pH 3), and added with the fresh reactants. Such 10 number of cycles yielded 81, 81, 80, 78, 67, 63, 59, 55, 52, and 51% of the SMX degradation. At the end of the 10th cycle, α-Cellulose-Fe3O4-CTNs-SH-Laccase ex- Furthermore, the repeated degradation of SMX by α-Cellulose-Fe3O4-CTNs-SH-Laccase was performed in a repeated cycle experiment ( Figure 10). The free laccase can access its application of SMX degradation in only one cycle. After completion of the first degradation cycle, α-Cellulose-Fe3O4-CTNs-SH-Laccase was retrieved by an external magnet, washed thoroughly with acetate buffer solution (100 mM, pH 3), and added with the fresh reactants. Such 10 number of cycles yielded 81, 81, 80, 78, 67, 63, 59, 55, 52, and 51% of the SMX degradation. At the end of the 10th cycle, α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase exhibited 51% of SMX degradation. These all results corroborated the potential of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase biocatalysts for environmental application and possibilities of developing a reactor scale treatment approach for decontamination of the pharmaceuticals contaminants. Thus, all biocatalysis and SMX degradation studies marked the importance of α-Cellulose-Fe 3 O 4 -CTNs-SH-Laccase catalysis.
hibited 51% of SMX degradation. These all results corroborated the potential of α-Cellulose-Fe3O4-CTNs-SH-Laccase biocatalysts for environmental application and possibilities of developing a reactor scale treatment approach for decontamination of the pharmaceuticals contaminants. Thus, all biocatalysis and SMX degradation studies marked the importance of α-Cellulose-Fe3O4-CTNs-SH-Laccase catalysis.

Conclusions
In conclusion, this study developed a new immobilization nano-support using nanobio-composite of paper-waste derived α-cellulose, Fe3O4 NPs, CTNs, and thiol (-SH) functionalization. The studied characterizations confirmed the successful synthesis of the α-Cellulose-Fe3O4-CTNs-SH-Laccase. The α-Cellulose-Fe3O4-CTNs-SH-Laccase was found to be an excellent biocatalyst with improved stabilities of thermal, pH, and storage. The reusability potential of α-Cellulose-Fe3O4-CTNs-SH-Laccase assured its potential for many laccase based applications. The significant potential of α-Cellulose-Fe3O4-CTNs-SH-Laccase was found in the decontamination of SMX from water. Thus, the developed biocatalyst α-Cellulose-Fe3O4-CTNs-SH-Laccase is novel, super-magnetic, waste-derived, and can be applied in the degradation of environmental pollutants.