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

Abstract

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.

1. 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 recycled fibers; however, for some other paper grades, such as cardboard or newspaper, this percentage can be close to about 99%, while achieving zero waste [7]. Therefore, sustainable practices to extract cellulosic fibers from waste wood, paper waste, and cardboard waste are suggested to compensate the principle called the “cellulose gap” include decrease dependence on wood cellulose and increase material reuse, and circular bio-economy practices [8,9,10].

Though cellulosic fibers share only a small stake in the manmade fiber sector, their unique properties related to moisture absorption [11], mechanical strength [12], biocompatibility, and some other functionalities renders cellulosic fibers are indispensable for various processes, products, and ultimately numerous technologies [13,14]. The consumption of cellulosic per capita is anticipated to be increasing constantly [1], but the cultivation of trees as the major source of cellulosic fibers have limited scope to expand [15,16,17]. Therefore, recycling cellulose from waste is generally considered as a more sustainable alternative from an environmental protection point of view [18], meanwhile, it allows the reuse of the cellulose fibers to produce new paper, cardboards, value-added materials (α-cellulose, cellulose nanofibers, nanocomposites) and, thus, minimize the consumption load on natural resources, and simultaneously reduces the generation of waste [19].

The term “circular economy” was predominantly developed by the Ellen MacArthur Foundation [20]. This can be archived by two major routes: Those from where biodegradable waste having nutrients are suitable to return to nature or reuse [21,22,23] and non-degradable waste needs cycling for biopolymers to reduce pollution, for the paper sector as well [24]. However, cellulose fiber processing from paper pulp involves different chemical–physical processes and specific operating conditions to facilitate the dissolution and regeneration of cellulose materials [25,26,27,28,29]. The extraction of cellulose from paper waste and its utilization has to be made evident to exert an excellent response on the circular economy projects worldwide with the involvement of sustainability [30]. Our group has recently reported a greener method for the production of α-cellulosic fibers from print paper waste and their utilization in the remediation of nanomaterial waste [31,32] and preparation of super-magnetic α-cellulose fiber-chitosan composite for covalent laccase immobilization [33].

An alternative method for enzyme immobilization using biocompatible nano-supports is an attractive approach for the improved stability and economic feasibility of potential biocatalysts [34,35,36]. The immobilization of laccase and exploring its environmental applications has received enormous attention due to ease of separation, long-term stability, and reuse of the immobilized biocatalysts [37,38]. Thiolation of the CTNs surfaces is an emerging methodology for enzymes, peptides, and proteins immobilization, as well as for developing potential biotechnological and environmental applications-degradation of metabolic disruptors as well [39,40,41,42]. Antibiotics used for livestock, fish farms, and humans are evolving as a serious pollutant in aquatic environments [43]. Their presence in numerous other environmental fragments including surfaces, soils, and groundwater, and some other biota is of great concern include environmental protection, antibiotic resistance, animal and human health [44,45]. Our group has reported a mechanism for thiolation of CTNs surfaces supported onto the magnetized halloysite nanotube and their application in the degradation of ampicillin and other organic compounds [46].

In this report, we propose immobilization of laccase enzyme using thiolation (–SH) mechanism of the amino (–NH₂) groups of the CTNs supported by magnetized-α-cellulose and further employed for degradation of sulfamethoxazole a potent metabolic disruptor. The magnetized nanocomposite of CTNs supported by α-cellulose was prepared, and further thiolated the (–NH₂) groups of CTNs, and applied effectively for immobilization laccase and degradation of the pharmaceutical compound sulfamethoxazole. Moreover, different factors including immobilization activity recovery, laccase loading capacity, the stability of pH, temperature, and storage, were first optimized. We performed the morphological and structural characterization of developed materials using independent techniques including high-resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR) analyses. The immobilized laccase enzyme in the biocompatible α-cellulose-Fe₃O₄-CTNs-SH-Laccase can be a novel efficient material for successfully treating water containing sulfamethoxazole antibiotics.

2. Materials and Methods

2.1. Materials

FeCl₂·4H₂O, FeCl₃·6H₂O, glutaraldehyde, and NH₃·H₂O were obtained from Daejung Chemicals, Sheung-si, Gyeonggi-do, South Korea. Chitosan (CTNs, low molecular weight), guaiacol (GUA, assay ≥ 98%), 2,2-Azino-bis(3 ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS, Liquid Substrate System), laccase from Trametes Versicolor (powder), N,N-dimethylformamide (99.8% DMF), thioglycolic acid (≥98%), N-hydroxysuccinimide (≥97.0% (titration) NHS), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (≥98% (titration), EDAC.HCl), and sulfamethoxazole (SMX) were obtained by the Sigma Aldrich, St. Louis, MO, USA.

2.2. Synthesis

The extraction of α-cellulose from paper waste biomass, the magnetization of extracted α-cellulose (α-Cellulose-Fe₃O₄), and CTNs loading over magnetized α-cellulose (α-Cellulose-Fe₃O₄-CTNs) was carried out as previous report [33]. The thiolation of α-Cellulose-Fe₃O₄-CTNs was carried out in the following steps. First, α-Cellulose-Fe₃O₄-CTNs was washed thoroughly with distilled water to remove impurities. This well washed 100 mg of α-Cellulose-Fe₃O₄-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-Fe₃O₄-CTNs was carried out. For this, NHS-ester (0.67 mL/L) was added to ultrasonicated 15 mL solution of α-Cellulose-Fe₃O₄-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-Fe₃O₄-CTNs (α-Cellulose-Fe₃O₄-CTNs-SH) were separated magnetically. The separated α-Cellulose-Fe₃O₄-CTNs-SH was washed thoroughly with Na-acetate buffer (pH 5.0 100 mM) several times. The well washed α-Cellulose-Fe₃O₄-CTNs-SH was further applied in laccase immobilization experiments.

2.3. Laccase Immobilization on α-Cellulose-Fe₃O₄-CTNs-SH

The laccase (1.5 mg/mL) immobilization on α-Cellulose-Fe₃O₄-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-Fe₃O₄-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-Fe₃O₄-CTNs-SH-Laccase (0.1 mL solution containing 1 mg of α-Cellulose-Fe₃O₄-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 as follows. The formulae of the measurements of activity recovery (%) were given by Equation (1):

$$Activity recovery (\%)=A_{IL}/A_{FL}\times 100$$

(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-Fe₃O₄-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):

$$Laccase loading (mg/g)=\left(C_{bi}-C_{ai}\right)V/W$$

(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-Fe₃O₄-CTNs-SH in gram (g). Furthermore, the relative activity is given by the following Formulae (3):

$$Relative activity (\%)=A_e/A_i \times 100$$

(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-Fe₃O₄-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-Fe₃O₄-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-Fe₃O₄-CTNs-SH-Laccase was carried out by incubating it at various pH (1–9) 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-Fe₃O₄-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-Fe₃O₄-CTNs-SH-Laccase was done. The reactants in first cycle include; substrate ABTS 1 mL (90 µM), α-Cellulose-Fe₃O₄-CTNs-SH-Laccase (0.1 mL solution containing 1 mg of α-Cellulose-Fe₃O₄-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-Fe₃O₄-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-Fe₃O₄-CTNs-SH-Laccase. The details of material characterizations were given in Supplementary Materials Method S1.

2.4. SMX Degradation by α-Cellulose-Fe₃O₄-CTNs-SH-Laccase

In the typical reaction mixture, SMX (25 ppm), α-Cellulose-Fe₃O₄-CTNs-SH-Laccase (0.1 mL solution containing 1 mg of α-Cellulose-Fe₃O₄-CTNs-SH-Laccase), redox-mediator GUA (500 µM) and sodium acetate buffer (100 mM, pH 3) was taken and kept shaking at 200 rpm, 30 °C for 20 h. After completion of the reaction, α-Cellulose-Fe₃O₄-CTNs-SH-Laccase separated magnetically, and retained solution analyzed spectrophotometrically at 287 nm to determine SMX degradation [47]. The optimization of reaction time for SMX degradation was done studying SMX degradation at 4, 8, 12, 16, 20, and 24 h. The pH and temperature optimum for SMX degradation by α-Cellulose-Fe₃O₄-CTNs-SH-Laccase study was carried out by varying the initial pH of reaction from 2–8 and temperature of 20, 30, 40, and 50 °C. The effect of shaking conditions was studied by carrying out a reaction at 50, 100, 150, 200, and 250 rpm. Finally, the repeated cycle degradation of SMX was carried by α-Cellulose-Fe₃O₄-CTNs-SH-Laccase. In this experiment, after completion of first degradation cycle, α-Cellulose-Fe₃O₄-CTNs-SH-Laccase separated magnetically, and fresh reactants were added for the second cycle. Similarly, 10 degradation cycles were carried out.

2.5. Statistical Analysis

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

3. Results and Discussion

3.1. 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-bio-catalyst α-Cellulose-Fe₃O₄-CTNs-SH-Laccase was further investigated the environmental application of SMX degradation (Figure 1).

3.2. Characterizations

3.2.1. HR-TEM Analysis

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 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 Fe₃O₄ NPs having narrow distribution, without any undesirable aggregation or agglomeration. Figure 2C also shows HAADF-STEM scan images of the carbon (Figure 2D), oxygen (Figure 2E), nitrogen (Figure 2F), Fe (Figure 2G), and sulfur (Figure 2I), for the selected area (Figure 2C). The elemental mapping results showed that the distribution of Fe and O in Fe₃O₄ were consistently validated. Furthermore, it was significant to reveal that the subsequent modification of the Fe₃O₄ 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-Fe₃O₄-CTNs-SH. This observation also confirmed the successful thiolation of α-Cellulose-Fe₃O₄-CTNs. As one can see in Figure 2J, α-Cellulose-Fe₃O₄-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 Fe₃O₄ loading, CTNs grafting, and thiolation grafted chitosan.

3.2.2. XRD, VSM, XPS, and FT-IR Analysis

Figure 3 displays the XRD patterns of composite α-Cellulose-Fe₃O₄-CTNs-SH. The XRD curve was approximately similar to that of Fe₃O₄ NPs [48], and there was no diffraction peak belonging to sulfur at 2θ = 20 to 70°, indicating that -SH or thiol is organic and amorphous. XRD results revealed the excellent stability of Fe₃O₄ NPs after the thiolation process performed for the immobilization of laccase enzymes. The Fe₃O₄ NPs diffraction peaks were assigned at 22.57°, 35.61, 43.39, 57.09, and 62.88° 2θ angles conforming to the crystalline planes for the (111), (311), (400), (511), and (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 Fe₃O₄ NPs, thus clear diffraction peaks appeared with strong intensity. Thus, the XRD analysis confirms successful loading and excellent stability of Fe₃O₄ NPs onto the α-cellulose surfaces.

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-Fe₃O₄-CTNs-SH was performed. Figure 4 gave VSM plot of the α-Cellulose-Fe₃O₄-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-Fe₃O₄-CTNs-SH.

XPS analysis was used to characterize the elemental profile of α-Cellulose-Fe₃O₄-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 -NH₂ of The CTNs and laccase. XPS spectra and elements confirm successful loading of the presence of Fe₃O₄ NPs and CTNs. Furthermore, the high-resolution spectra of Fe 2p were performed for α-Cellulose-Fe₃O₄-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 Fe₃O₄ NPs [50]. Since Fe 2p curve fitting analysis indicated the presence of both the oxidation states of Fe²⁺ and Fe³⁺ [51]. The XPS results revealed that Fe₃O₄ was remained stable onto the α-cellulose ever after modified using CTNs and laccase immobilization.

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.

3.3. Laccase Immobilization Studies

3.3.1. 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 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, 1.2, 1.5, 1.8, and 2.1 mg/mL, were found to be 66.22, 73.85, 79.76, 88.69, 99.16, 94.86, and 93.28%, respectively (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.

3.3.2. Stability Studies of Immobilized Laccase

The immobilization strategy provides several advancements for enzyme biocatalysis [34,37]. This mainly includes temperature stability, pH stability, and storage stability compared to the free laccase. The temperature stability of α-Cellulose-Fe₃O₄-CTNs-SH-Laccase was explained in Figure 8A. The α-Cellulose-Fe₃O₄-CTNs-SH-Laccase exhibited relative activity (%) of 98, 97, 94, 90, 85, and 81%, at 30, 60, 90, 120, 150, and 180 min of the incubation at temperature 60 °C, respectively (Figure 8A). However, free laccase possessed 90, 85, 81, 74, 69, and 65% of relative activity, at 30, 60, 90, 120, 150, and 180 min of the incubation at 60 °C temperature. The obtained data suggest that the thermal stability of immobilized laccase enhanced significantly. The binding of the enzyme to support provides additional support for the enzyme to enhance thermal stabilities [37].

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-Fe₃O₄-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-Fe₃O₄-CTNs-SH. Additionally, the microenvironment of α-Cellulose-Fe₃O₄-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-Fe₃O₄-CTNs-SH-Laccase displayed improved storage stability over free laccase. Therefore, all the stability analysis (Figure 8A–C) of α-Cellulose-Fe₃O₄-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.

3.3.3. 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-Fe₃O₄-CTNs-SH and applied in the 10 repeated cycles (Figure 8D). The α-Cellulose-Fe₃O₄-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-Fe₃O₄-CTNs-SH-Laccase as a novel, cost-effective, and environmental-friendly biocatalyst for laccase based applications.

By considering the valuable materials like α-Cellulose-Fe₃O₄-CTNs, and its application in the development of new enzyme immobilization techniques were highly essential. In previous work, laccase immobilization on α-Cellulose-Fe₃O₄-CTNs was done by cross-linking agent glutaraldehyde (GTA) [33]. Therefore, we compared the obtained results of this study with “GTA modified α-Cellulose-Fe₃O₄-CTNs” for laccase immobilization. The thiol modified α-Cellulose-Fe₃O₄-CTNs (α-Cellulose-Fe₃O₄-CTNs-SH) gave a significantly higher capacity of the laccase loading (169 mg/g) than the GTA modified α-Cellulose-Fe₃O₄-CTNs (73.30 mg/g) [33]. Similarly, the stability of temperature was improved with α-Cellulose-Fe3O4-CTNs-SH-Laccase [33]. Looking at the obtained results, thiol modified α-Cellulose-Fe₃O₄-CTNs were found to be an excellent material for enzyme immobilization strategy.

3.4. 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). At the 4, 8, 12, 16, 20, and 24 h, α-Cellulose-Fe₃O₄-CTNs-SH-Laccase gave 35, 48, 63, 70, 83, and 82% degradation of the SMX, respectively (Figure 9A). The increase in incubation time of SMX with α-Cellulose-Fe₃O₄-CTNs-SH-Laccase increases the degradation of SMX until 20 h. After 20 h, SMX degradation remains constant. Hence, the optimum time for SMX degradation was found to be 20 h. Further, the effect of pH on SMX degradation was evaluated (Figure 9B). At the pH of 2, 3, 4, 5, 6, 7, 8, and 9, α-Cellulose-Fe₃O₄-CTNs-SH-Laccase gave 82, 83, 81, 60, 51, 44, 16, and 5% degradation, respectively. The higher SMX degradation was observed at the acidic pH range 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 exhibited 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.

4. Conclusions

In conclusion, this study developed a new immobilization nano-support using nano-bio-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 bio-catalyst α-Cellulose-Fe₃O₄-CTNs-SH-Laccase is novel, super-magnetic, waste-derived, and can be applied in the degradation of environmental pollutants.