Immobilization of laccase on magnetic cellulose beads for enhanced biodegradation of bisphenol A and a metal-complex dye

Immobilization of laccase on magnetic cellulose beads for enhanced biodegradation of bisphenol A and a metal-complex dye | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immobilization of laccase on magnetic cellulose beads for enhanced biodegradation of bisphenol A and a metal-complex dye Gulay Bayramoglu, Mehmet Yakup Arica This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8013262/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Cellulose-based materials can be an ideal immobilizing support for enzymes due to their abundance in nature, non-toxicity, sustainability, and biodegradability. Furthermore, they do not leave any toxic residue behind after the process is utilized. In this work, magnetic cellulose beads (MCB) were coated with polydopamine-polyethyleneimine (PDA-HMDA). The MCB@PDA-HDMA beads were then functionalized by chelating Cu(II) ions or reacting with glutaraldehyde (GA) for the immobilization of laccase via metal chelate interaction or covalent binding, respectively. PDA-HMDA polymer pairs were coated onto MCB to enhance Cu(II) ions chelation and GA functionalization, which provided metal chelate interaction and covalent binding sites for laccase. The MCB@PDA-HDMA beads-based preparations were characterized using FT-IR, X-ray diffraction, SEM, TEM, and VSM. The amounts of immobilized laccase via metal chelate interaction, covalent binding, and adsorption on the MCB@PDA-HDMA-Cu(II)-Lac, MCB@PDA-HDMA-GA-Lac, and MCB@PDA-HDMA beads were found to be 87.9, 51.6, and 42.4 mg/g beads, respectively. The highest activity yield order of immobilized laccase preparations was MCB@PDA-HDMA-Cu(II)-Lac (81.6%) > MCB@PDA-HDMA-GA-Lac (68.4%) > MCB@PDA-HDMA (48.7%) compared to the free laccase using syringaldazine as an artificial substrate. The free enzyme, MCB@PDA-HDMA-Cu(II)-Lac, MCB@PDA-HDMA-Lac preparations exhibited their maximum activities at pH 6.0, whereas MCB@PDA-HDMA-GA-Lac showed its maximum activity at 6.5. The maximum activity for all the laccase preparations was obtained at 35°C. Furthermore, the immobilized forms of laccase displayed good performance over a broader pH range and at higher temperatures. The free laccase was wholly inactivated at 70°C after 60 min incubation in the substrate-free medium, while the MCB@PDA-HDMA-GA-Lac > MCB@PDA-HDMA-Cu(II)-Lac > MCB@PDA-HDMA preserved about 44.6, 21.3, and 19.4% of their initial activities, respectively. The biodegradation of two model pollutants, Bisphenol A (BPA) and Reactive Green 5 (RG-5, metal complex dye), with the free enzyme and MCB@PDA-HDMA-Cu(II)-Lac preparation was studied batch vise. In the presence of acetosyringone as a mediator compound in the reaction medium, biodegradation amounts of BPA (at 20 mg/L) and RG-5 dye (10 mg/mL) were detected as 96.9% and 78.2%, respectively, using MCB@PDA-HDMA-Cu(II)-Lac preparation for a 120 min reaction time. The MCB@PDA-HDMA-Cu(II)-Lac preparation displayed high biodegradation performance for both tested pollutants compared to the free laccase. Moreover, the MCB@PDA-HDMA-Cu(II)-Lac and MCB@PDA-HDMA-GA-Lac preparations were used for the degradation of BPA and RG-5 in a batch system over five consecutive cycles. These obtained results make the immobilized laccase preparations favorable candidates for many environmental applications, such as wastewaters management and remediation, where improved enzyme performance and reusability are key parameters. Magnetic particles Cellulose beads Dopamine-polyethyleneimine coating Laccase Phenolic compounds Enzymatic degradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The biodegradation of pollutants from wastewaters using industrially important enzymes can be achieved through a few reaction steps under mild reaction conditions, resulting in reduced waste. Considering the important roles of enzymatic reactions, the high cost, low stability, and limited reuse numbers of free enzymes limit their extensive applications in biotechnological areas (Antanaskovic et al., 2024 ; Arica et al., 2017 ; Bayramoglu and Arica, 2021 ; Borges et al., 2024 ). The fixation of enzymes on insoluble supports can significantly reduce the limitations mentioned above by enhancing their operational stability under various experimental conditions, thereby dominating the enzymatic reaction and permitting consecutive operations (Abkenar and Dehnavi, 2025 ; Bayramoglu et al., 2019 ; Borham et al.,2025; Garg et al ., 2024; Hoang and Kim, 2025 ; Maine et al., 2025 ). The choice of support and method for enzyme immobilization is crucial for preserving the activity of immobilized enzymes for biotechnological applications. Various organic and inorganic support materials, including cellulose, chitosan, poly(acrylic) polymers, polymeric resins, clay minerals, magnetic particles, and silica nanoparticles, have been utilized for the immobilization of enzymes (Hussain et al., 2025 ; Ishak et al., 2025 ; Bayramoglu et al., 2018 ; El-Shishtawy et al., 2025 ; Gurgel et al., 2025 ). Most of the enzymes on these support materials have been immobilized via chemical and physical methods. The former is the most widely employed method, which generates a covalent linkage between enzymes and the support (Alokpa et al., 2025 ; Kim et al., 2017 ; Li et al., Lu et al., 2025 ; Souza et al., 2025 ). This method provides many advantages, such as minimal enzyme leakage from support, a high amount of enzyme loading, repeated usability, and improving thermal and pH stabilities of enzymes compared to the physical immobilization method (Moayedi and Yousefi, 2025 ; Mota and Gimenez, 2023 ; Oraby et al., 2025 ; Othman and Flaifil, 2025 ). Particularly, supports materials with pendant amine groups can be activated with various bifunctional agents, such as glutaraldehyde and carbodiimide, to react and form a covalent linkage with different groups on the surface of the enzyme molecules, including amino, hydroxyl, and thiol groups. These linkages can provide negligible deformation to the three-dimensional structure with minimal chemical influence on the structure of enzymes (Bayramoglu et al., 2013 ; Lin et al., 2025 ; Feng et al., 2025 ). Furthermore, glutaraldehyde-activated supports can be practically ideal for the covalent immobilization of enzymes on both laboratory and industrial scales (Bayramoglu et al., 2018 ; Xu et al., 2024 ). Glutaraldehyde-activated supports can react with enzymes under mild reaction conditions and over a wider pH range, allowing for facile covalent interactions. Moreover, the bond formed between the enzyme and the supports is very stable under wet storage conditions. Therefore, glutaraldehyde-activated supports have been widely used for the immobilization of proteins, such as glutaraldehyde-activated acrylic polymers, polyethyleneimine-modified various microbeads (Bayramoglu et al., 2017 ; Khanam et al., 2024 ), microspheres containing amine groups, and magnetic nanoparticles, which have been utilized in earlier enzyme immobilization studies. Cellulose beads can be prepared with diameters ranging from micro to millimeters and used in a variety of complex applications, such as chromatographic fields, enzyme immobilization technology, and drug delivery systems. Cellulose and its derivatives possess several essential properties that make them suitable for the applications above, including sustainability, non-toxicity, biocompatibility, biodegradability, and good mechanical properties (Bayramoglu et al., 2024 ; Li et al., 2024 ; Sourgi and Dehnavi, 2025 ; Liu et al., 2025 ). Cellulose is made of D-glucopyranosyl units and linked with each other via β-1.4-glycosidic bonds, and each sugar unit has three available hydroxyl groups on C2, C3, and C6 (Bao et al., 2022 ; Oktaviani et al., 2024 ; Udoetoka et al., 2016 ). Particularly, cellulose-based materials have been highly recommended as an ideal carrier for enzyme immobilization (Xing et al., 2021 ; Yan and Hou, 2025 ). Cellulose-based materials have garnered important attention as enzyme immobilization materials due to their distinctive structural properties, which include an interior porous assembly, a large surface area, and high numbers hydroxyl groups that are easily chemically variable (Lin et al., 2025 ). The hydroxyl groups on cellulose fibers have not formed easily covalent bonds with enzymes; chemical alteration is necessary to present useful groups, such as amino groups, to simplify covalent immobilization. Enzymes can be covalently immobilized on cellulose fibers to enhance enzymatic efficiency and production yields, while also contributing to green technology and sustainable sources. Many enzymes have been immobilized on cellulose-based materials, including laccase, trypsin, lipase, glucose oxidase, peroxidase, and tyrosinase (Kim et al., 2017 ; Mota and Gimenez, 2023 ; Sathishkumara et al., 2014 ; Yang et al., 2025 ; Yu et al., 2025 ), among others. Moreover, cellulose-based magnetic supports can permit easy stop of the reaction using an external magnet, and also provide easy reusability. Laccase EC 1.10.3.2) is an oxidation–reduction enzyme and produced by many plants and fungi, and containing a copper atom in its catalytic center. Laccase is capable of degrading numerous phenolic compounds, including various dyes, phenols, and complex aromatic compounds. The critical limitation of the use of laccase in pollutant biodegradation is low stability and short persistence of the free enzyme, as well as the high production cost of the enzyme (Aghera et al., 2025 ; Al- Sareji et al., 2023; Bayramoglu et al., 2012 ; Petrila et al., 2025 ; Shen et al., 2025 ). These important problems can be eliminated by immobilization, thereby increasing their economic value (De Paula et al., 2025 ; Jin et al., 2025; Kamal et al., 2025 ). An efficient immobilization approach for laccase relies on the constitution and performance of the support, as well as the choice of immobilization method. Among numerous immobilization protocols, covalent coupling is a desired method, as it allows the enzyme to be tightly bound to the support (Kokar et al., 2024 ; Kumar and Sridhar, 2024 ; Kyomuhimbo and Brink, 2023 ). In this work, laccase from a white rot fungus ( Trametes versicolor ) was immobilized on the PDA-HMDA grafted magnetic cellulose beads (i.e., MCB@PDA-HDMA) either through metal chelate interaction or glutaraldehyde coupling. For this, the magnetic cellulose beads were synthesized and coated with PDA in the presence of HMDA. In the second step, for immobilization via metal chelate interaction, Cu(II) ions were chelated onto the beads, or the beads were activated with GA for covalent attachment. Characterization of the immobilized enzyme preparations was investigated under different experiments using free laccase as a control system. The optimum pH and temperature, kinetic parameters, stability, and reusability of both free and immobilized enzymes were studied. The immobilized enzyme preparations were used to degrade BPA and a metal dye, RG-5, from an aqueous medium. Results showed that the performance of the immobilized laccase preparations in degrading BPA and RG-5 dye was very effective; hence, the presented immobilized methods could provide a new approach to removing phenolic compounds from industrial effluents. To the best of our knowledge, the presented laccase immobilized methods and RG-5 degradation have not been reported. Also, the support materials as prepared were not used for any enzyme immobilization. Materials and Methods 2.1. Materials Laccase (EC 1.10.3.2, 20 U/mg solid) from Trametes versicolor, 4-hydroxy-3,5-dimethoxyhydroxybenzaldehyde (syringaldazine), microcrystalline cellulose (20–160 µm), dopamine, Tris-HCl, hexamethylenediamine, glutaraldehyde solution (25 %), Bisphenol A, Reactive Green 5 (Procion Green H 4G), and sodium hydroxide were supplied from Sigma-Aldrich Chem. Co, Germany. 2.2. Synthesis of Fe 3 O 4 particles Fe 3 O 4 particles were synthesized via thermal precipitation of Fe(III) and Fe(II) as reported earlier (Arica et al., 2017). Briefly, 200 mL of FeCl 3 (0.3 mol/L) and 100 mL of ethylene glycol were added to a reaction vessel and magnetically agitated for approximately 10 min. Then, 200 mL of FeCl 2 (0.1 mol/L) and polyethylene glycol (PEG, 6000; 2.0%) mixture was transferred into the reaction medium and agitated for an additional 30 min. After this period, 50 mL of ammonia solution (25%, v/v) was added to the reaction vessel and refluxed at 70 °C for 2.0 h, followed by an additional 1.0 h at 90 °C. The magnetic particles were dried under reduced pressure at 25 °C. 2.3. Preparation of MSB and grafting with PDA-HMDA MCB were prepared via the extrusion-dropping method as described in an earlier work (Bayramoglu et al., 2024). An alkaline solution was prepared by adding 14 g of NaOH and 24 g of urea to 162 mL of H₂O and stirring the mixture magnetically at 15 ºC for 30 min. Then, 6.0 g of cellulose microcrystalline powder and 4.0 g of magnetic nanoparticles were transferred to the medium, and further stirred at 250 rpm for 60 min. The resulting blend was dropped into the sodium chloride solution (400 mL, 15%) using a syringe pump. The prepared MCB was collected using an external magnet and cleaned with deionized water. The dopamine molecule has catechol and amine functional groups, and under alkaline conditions, dopamine self-polymerizes to form PDA and generates coating layers on the surface of different organic and/or inorganic materials. PDA has several distinctive possessions, including biocompatibility, strong adhesion, and antioxidant properties. For PDA/HMDA coating, the magnetic cellulose beads (2.0 g) were transferred to a reaction vessel containing 99 mL of Tris-HCl buffer (pH 8.5). Then, 600 mg of dopamine and 1.0 mL of HMDA were added, and stirred magnetically for 10 min. After that, the reaction vessel was placed on an orbital shaker and the grafting reaction was performed at 50 ºC for 18.0 h. After the reaction period, the MCB@PDA-HMDA beads were cleaned with distilled water to remove any unreacted dopamine and HMDA. Next, the MCB@PDA-HMDA (2.0 g) was transferred to a glutaraldehyde (GA) solution (100 mL, 1.0%) to produce functionalized magnetic cellulose beads (MCB@PDA-HMDA-GA). The activation reaction was carried out at 50 ºC while continuously agitating for 4.0 h. The MCB@PDA-GA were then washed with an acetic acid solution (1.0%, v/v) and subsequently with deionized water to remove any reaction impurities. Some of the MCB@PDA-HMDA beads were chelated with Cu(II) ions for immobilization of laccase via metal-chelate interaction. For this, Cu(II) solution (100 ppm) was prepared from CuCl 2 in deionized water at a pH of 4.5. The MCB@PDA-HMDA beads (4.0 g) were transferred to the Cu(II) solution (100 mL) and stirred at 100 rpm and at 25 °C for 2.0 h. Then, the Cu(II) ions chelated MCB@PDA-HMDA beads were washed with phosphate buffer solution (50 mM, pH 6.5). The initial and final concentrations of Cu(II) ions in the medium were determined using a flame atomic absorption spectrophotometer (AAS, Shimadzu AA6800, Japan). At least 10 AAS measurements were recorded, and the mean of the data was used. The amount of chelated Cu(II) ions (μmol/g beads) was determined as the change in the before and after contacted he beads Cu(II) ions solution. The leakage of the chelated Cu(II) ions from the beads was examined in the pH range of 5.0–9.0. The MCB@PDA-HMDA-Cu(II) beads were incubated with Cu(II) ion solution at different pH values while stirring at 25 ºC for 12 h, and the released Cu(II) ion concentration was detected in the supernatants using AAS as described above. Subsequently, MCB@PDA-HMDA-GA and MCB@PDA-HMDA-Cu(II) beads were utilized for laccase immobilization, with MCB@PDA-HMDA beads serving as a control system. The preparation step of support materials is schematically presented in Figure 1. 2.4. Immobilization of laccase via adsorption and metal chelate interaction The immobilization of laccase on MCB@PDA-HMDA-Cu(II) was studied based on the incorporation of borderline Cu(II) ions on the chelating groups of MCB@PDA-HMDA functionality, and also MCB@PDA-HMDA was used as a control system. Immobilization of laccase on the MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II) beads was realized at different pH values, in either acetate (100 mmol/L, pH 4.0–5.0) or in phosphate buffer (100 mmol/L, pH 6.0–9.0). The initial laccase concentration was 2.0 mg/mL in the corresponding buffer solution, and immobilization of laccase via adsorption and metal chelate interaction was performed at 17 °C while stirring for 2.0 h. Then, the laccase immobilized on both magnetic beads was collected magnetically from the medium, and cleaned with the corresponding buffer solution. The amount of protein in the solutions was determined by the Bradford method (1976) as described previously. To determine the reusability of MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II) beads, the laccase adsorption/desorption round was repeated five times by using the same support materials. The laccase desorption from the MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II) beads was achieved using a KSCN solution as the desorption agent (10 mL, 1.0 mol/L, pH 8.0). The laccase-laden MCB@PDA-HMDA or MCB@PDA-HMDA-Cu(II) beads were added in the desorption medium and stirred magnetically at 150 rpm and at room temperature for 2.0 h. The samples were cleaned, then reutilized in the next run for immobilization of laccase. 2.5 Immobilization of laccase via covalent attachment The glutaraldehyde-activated beads (i.e., MCB@PDA-HMDA-GA, 1.0 g) were transferred to 2.0 mg/mL of laccase solution (10.0 mL, pH 8.0, 15 ºC) and mixed for 4.0 h in a rotated incubator shaker. Then, the laccase-immobilized beads (i.e., MCBs@PDA-HMDA-Lac) were cleaned with phosphate buffer solution (50 mM, pH 6.5). The amount of protein in the solutions was determined at 595 nm using a UV/Vis spectrophotometer (PG Instruments Ltd., Model T80 +, PRC). A BSA standard curve was used to calculate the protein amount in the solutions. 2.6. Activity assays of laccase preparations and kinetic parameters The laccase activity assays were studied over the pH range of 3.0–8.0 at 25 °C, as well as at temperatures between 15 and 60 °C and at pH 6.0. In these experiments, the laccase activity was measured the oxidation rate of syringaldazine at 530 nm as reported earlier (Bayramoglu et al., 2019). The relative activities of the laccase preparations were considered as the ratio between the measured activities at the tested pH or temperature and the highest level of activity. The K m and V max values of the laccase preparations were determined using different concentrations (0.1-2.0 mmol/L) of syringaldazine as substrate in acetate buffer at pH 6.0 and at 25°C. The kinetic parameters of the enzyme preparations were calculated using the Michaelis-Menten equations. 2.7. Thermal and storage stabilities of the laccase preparations The thermal and storage stabilities of the enzyme preparations were evaluated as reported previously (Bayramoglu et al., 2019). The thermal stabilities of laccase preparations were detected after incubation at various temperatures (15–65 ºC) in phosphate buffer at pH 6.0 for 120 minutes. The storage stability of the laccase preparations was measured at one-week intervals over an 8-week period while storing at 4 ◦C, and the initial activity was defined as 100%. The reusability of laccase preparations was determined by measuring the activity of the same sample five times, and each experiment was triplicated. 2.8. Degradation of Bisphenol A and Reactive Green 5 with laccase preparations The reaction mixture containing BPA and RG-5 dye (each 50 mg/L) in phosphate buffer solution (pH 6.0, 50 mM, 5.0 mL) or adding acetosyringone (0.2 mM) in the same buffer. The laccase preparations in BPA or RG-5 solutions were incubated on an orbital shaker at 25 °C and 100 rpm for 120 min. Then, the reduction in the absorbance value of the BPA and RG-5 dye-containing medium was measured at 276 and 670 nm, respectively, using a UV/visible spectrophotometer. The percentage of the removed pollutant was calculated as: Removal pollutant (%) = [( C 0 − C ) /C 0 ] × 100 (1) where C 0 and C are the concentration of BPA or RG-5 dye in the initial medium and after time t (mg/L). After a given time period, a sample was removed and measured spectrophotometrically. 2.9. MALDI-ToF- MS studies The MALDI-ToF-MS analysis was performed as described previously (Bayramoglu et al., 2019). The mass spectra were achieved on a MALDI-ToF mass spectrometer (Voyager-DE™ PRO, Applied Biosystem, USA) at 337 nm. The spectra were obtained in positive-ion and linear mode, with an average of 100 shots. 2.10. Eco-toxicity studies Chlorella vulgaris was used in the algal growth inhibition tests according to the OECD guideline 201 (O.E.C.D. 2011). The growth rates of C. vulgaris were obtained at different BPA and/or RG-5 dye concentrations in the range of 5.0-25 mg/L for a 5-day period. The same test was also performed after 120 min of enzymatic treatment of the BPA and RG-5 dye solutions. The initial algal cell density of the medium was approximately 1.0 × 10 5 cells/mL, and cell counts were measured spectrophotometrically at 685 nm. Control groups were also included in the absence of BPA and RG-5, and were used for evaluating the results of ecotoxicological experiments. The immobilization test of Daphnia magna was also conducted using neonates (less than 24 hours old) in accordance with the guidelines of OECD No. 202 (OECD, 2004). The concentrations of BPA or RG-5 were 2, 5, 10, 15, 20, and 25 mg/L. Briefly, ten daphnids were transferred to 25 mL medium containing various concentrations of BPA or RG-5. The immobilization rates were calculated after 48 h of exposure. Each treatment test was triplicated. 2.11. Characterization of modified cellulose-based supports The available surface amine groups of the MCB@PDA-HMDA beads were determined with a potentiometric titration method. For this, 0.5 g of MCB@PDA-HMDA beads were added to the HCl solution (0.1 mol/L, 10 mL) and incubated in a rotary shaker at 25 ºC for 4.0 h. Then, the final concentration of HCl in the medium was measured using NaOH solution (0.05 mol/L). The specific surface areas of MCB and MCB@PDA-HMDA were determined by the BET method (Brunauer, Emmett, and Teller) using BET Surface Area Analyzers (Quantachrome Nova 2200 E, USA). Former to analysis, samples were incubated at 110 °C for 18 h under reduced pressure. The specific surface areas of the samples were determined. The FTIR spectra of the MNP, MCB, MCB@PDA-HMDA, and MCB@PDA-HMDA-Lac beads were obtained using a Nicolet TM ISTM 50 FTIR spectrometer (Thermo Fisher Scientific, USA). The surface morphologies of the MCB and MCB@PDA-HMDA beads were obtained using a scanning electron microscope (JEOL, Model JSM-5600, Tokyo, Japan). The transmission electron micrograph (TEM) images of the magnetic nanoparticles (MNP) were attained at an accelerating voltage of 120 kV using a JEM-1400 Plus electron microscope in TEM mode (JEOL Ltd, Akishima, Tokyo, Japan). The magnetic properties of the MNP, MCB, MCB@PDA-HMDA, and MCBs@PDA-HMDA were studied at room temperature using a vibratory sampling magnetometer (VSM; Model 155, Digital Measurement System Inc., Westwood, MA, USA). The X-ray diffraction (XRD) patterns of the MNP and MCBs@PDA-HMDA-Lac beads were obtained using Cu-Kα radiation, between 2θ of 20 and 80º, with a 0.1 increment and 2º min −1 scan speed (MiniFlex 600, Rigaku). Results and Discussions 3.1. Characterization of the as-prepared cellulose‑based support materials The amino group content of MCB@PDA-HMDA beads was determined to be 1.28 mmol/g. The specific surface areas of MCB and MCB@PDA-HMDA beads were determined as 27.8 and 19.7 m 2 /g, respectively. The results exhibited that the specific surface area decreased after grafting of MCB with PDA-HMDA polymers. The resduce in the surface area of the MCB beads after grafting with PDA-HMDA can result from the lessening in the pore sizes and pore volume upon integration of PDA-HMDA polymer molecules on the surface of the MCB beads. The ATR-FTIR spectra of pure cellulose, Fe 3 O 4 , MCB, MCB@PDA-HMDA, MCB@PDA-HMDA-Lac, and @PDA-HMDA-Cu(II)-Lac are displayed in Figure S1. For the pure cellulose, at 3338 cm −1 , an extensive band was observed for the stretching vibration of -OH groups of cellulose (Figure S1A). Two peaks at 2892 and 1640 cm −1 were seen due to stretching and vibration of C-H and C–C of pure cellulose, respectively. As seen in Figure S1B, a strong peak for the Fe-O bond was observed at 558 cm -1 , whereas the peaks at 1628 cm -1 and 3285 cm -1 could be characteristic of hydroxyl groups associated with H 2 O existence on the surface of the micro particles. The FTIR spectrum of MCB was similar to that of pure cellulose except that a wide adsorption band at 3452 cm -1 was observed (Figure S1C). This band was related to the stretching vibration of the –OH groups of cellulose and Fe-OH. The MCB@PDA-HMDA material was also examined using ATR-FTIR to confirm the PDA-HMDA grafted on the surfaces of MCB. As given in Figure S1D, the FTIR spectrum of the PDA-HMDA grafted beads displaying a comparatively extensive band in the 3305 cm -1 could be attributed to the stretching vibration of hydroxyl groups of cellulose/Fe-OH, and also amine groups of chitosan/HMDA. The observed peak at 1054 cm -1 could be due to the –NH shearing vibration of the amide groups. This observation could be attributed to the hydrogen bonds between the PDA and HMDA molecules. These observations showed that PDA-HMDA effectively grafted onto the MCB. All these detected variations were associated with the grafting of PDA-HMDA on the MCB beads. The FTIR spectra obtained after the immobilization of laccase showed significant changes in the vibration bands in the 1050–1650 cm -1 region, indicating that the enzyme was immobilized on the MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-Lac beads (Figure S1E and S1F, respectively). The observed peaks from the FTIR spectrum of the MCB@PDA-HMDA-Cu(II)-Lac beads at 1552 cm -1 and 1411 cm -1 originated from the immobilized laccase. Furthermore, the peak at 1634 cm -1 could be due to the stretching vibration of carbonyl (–C=O) groups. The peaks were observed at 1552 cm -1 and between 2897 cm -1 and 3326 cm -1 , which could be attributed to the N–H bending vibration (Figure S1E). Additionally, the peak at 3446 cm -1 was raised to the –NH stretching at resonance. The observation of these characteristic peaks on the MCB@PDA-HMDA-Cu(II)-Lac beads corroborates the successful immobilization of laccase via adsorption. As observed in Figure S1F, the reduction in peak extent at 3320 cm -1 indicates that laccase was successfully immobilized on MCB@PDA-HMDA-Lac. These results also demonstrated that laccase was effectively immobilized on the MCB@PDA-HMDA beads (Figure S1F). Figure S2 shows SEM images of the dry MCB@PDA-HMDA beads at two different magnifications. As seen in this figure, the MCB@PDA-HMDA beads were spherical in geometry and had an irregular surface appearance (Figure S2A). This could be related to the presence of MNP in the bead formulation. The presence of rough surfaces on the beads can provide high enzyme immobilization capacity, and also increase the surface area for the enzymatic reaction (Figure S2B). The TEM nanograph of the Fe 2 O 3 nanoparticles in the dry state is presented in Figure 2A. As shown in the figure, the Fe 3 O 4 nanoparticles had a nearly spherical shape with a diameter of approximately 12 nm. The photographs of the pure cellulose and MNP-entrapped cellulose beads are presented in Figure 2B. The pure cellulose beads were white in color, whereas the MCB@PDA-HMDA beads were black due to the existence of MNP, signifying that Fe 3 O 4 nanoparticles were uniformly entrapped in the cellulose beads. The average size of the PDA-HMDA grafted cellulose beads was around 1.2±0.13 mm (Figure 2B). The MCB@PDA-HMDA beads were simply removed from the reaction medium using an external magnet, demonstrating the sensitivity of the as-prepared magnetic beads to the applied magnetic force (Figure 2C). The magnetization data of the samples are presented in Figure S3. The saturation magnetization of the as-prepared samples was found to be 56.4, 32.6, and 29.3 emu/g for the MCB@PDA-HMDA-Lac beads, respectively. It was observed that saturation magnetization highly relies on the total amount of the MNP in the beads and directly influences the magnetic properties of the materials. As shown in this figure, the magnetization curves of the beads exhibited zero refraction tendency, indicating superparamagnetic properties. The presented data exhibited that the MCB@PDA-HMDA-Lac beads can be simply collected from the solution using an external magnet. The superparamagnetic nanoparticles incorporated into the beads were well characterized, and the targeted success was achieved; the MCB@PDA-HMDA-Lac beads did not clump together due to interactions with each other. Furthermore, the prepared magnetic beads were very stable, and the magnetic properties of the as-prepared support were sufficient. Thus, the support material was easily removed magnetically from the reaction medium within a short time and could be simply re-dispersed within a few seconds. In XRD analyses, the diffraction peaks of the Fe 3 O 4 nanoparticles within the MCB@PDA-HMDA-GA-Lac beads were completely observed. These data indicate that the construction of the Fe 3 O 4 was wholly well-preserved in the MCB@PDA-HMDA-GA-Lac beads during the preparation reactions (Figure S4). As shown in this figure, the MCB@PDA-HMDA-GA-Lac beads exhibit the similar diffraction peaks as the MNP, demonstrating that the magnetic properties were well-preserved. These data indicate that the magnetic properties of the MNP remained unchanged during the preparation step of the support. 3.2. Immobilization of laccase on the MCB@PDA-HMDA-Cu(II) via metal-chelate interaction and MCB@PDA-HMDA beads via adsorption The amount of immobilized laccase via metal chelate interaction with Cu(II) ions and adsorption was determined using the Bradford method (1976). The influence of the initial enzyme concentration on the adsorption performance of the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads was studied at pH 6.0 and 25 ºC for 2.0 h, and the enzyme concentrations were varied in the range 0.1-3.0 mg/mL in the medium. The amount of adsorbed laccase augmented with increasing initial concentration of the enzyme in the medium. The maximum immobilization capacities were found to be 87.9 and 42.4 mg/g for MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads, and the amounts of immobilized laccase per unit mass of the both support reached a plateau value at 2.0 mg enzyme/mL, and the retained laccase activity were found to be 81.6 %, and 48.7 comparable with the related literature, respectively (Bayramoglu et al., 2019). As shown in Figure 3A, the lowest amount of laccase was immobilized on the MCB@PDA-HMDA beads via the adsorption method compared to the metal chelate interaction on the MCB@PDA-HMDA-Cu(II). This could be resulted from the coordination complex of Cu(II) ions with the amino acid residues on the surface of the enzyme. The most important amino acid residues on the surface of laccase enzymes are the imidazole group of histidine, thiol group of cysteine, amino groups of lysine and arginine, and carboxyl groups of aspartic acid and glutamic acid, and these residues can make metal chelate interactions with Cu(II) ions. As reported earlier, an appropriate amount of Cu(II) ions preserves the three-dimensional active conformational structure of laccase, allowing it to bind to the substrate and undergo oxidation, thereby enhancing its activity (Bayramoglu et al., 2012; Jing et al., 2025; Li et al., 2024). Immobilization of laccase via Cu(II) ions interaction onto the surface of the MCB@PDA-HMDA-Cu(II) beads has enhanced the immobilization efficiency and enzyme activity compared to immobilization via adsorption (i.e., MCB@PDA-HMDA-Lac) to a certain level (Bayramoglu et al., 2012; Plesner et al., 2025; Ranimol and Sunkar 2022). The activity of the MCB@PDA-HMDA-Cu(II)-Lac was remarkably higher than that of the MCB@PDA-HMDA-Lac preparation. This result could be due to the presence of Cu(II) ions on the support, which affected the laccase activity. Because laccase enzyme is a multi-copper oxidase enzyme and needs copper ions to preserve its activity (Plesner et al., 2025). As reported in earlier studies (Bayramoglu et al., 2012), laccases require copper ions to maintain their active conformational structure, bind to their substrate, and facilitate electron transfer. Therefore, immobilization of laccase with Cu(II) chelate formation was also considered in this work. The influence of pH on the laccase immobilization efficacy and performance on the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads was studied in the pH range of 4.0-9.0 at 25 ºC with an initial laccase concentration 1.0 mg/mL. The maximum amount of enzyme binding was observed at pH 6.5 for both supports. As presented in Figure 3B, the laccase immobilization capacities of the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads were 75.1 and 33.8 mg/g beads, respectively. The Langmuir isotherm model equation was used to analyze experimental data. The isotherm equation is: q e = q m C e / (K d +C e ) (2) The experimental adsorption isotherm data is shown in Figure 3A. The isotherm model parameters q m , and K d values were calculated and presented in Table 1. The q m and K d values of immobilized laccases were 92.6 and 49.9 mg protein/g and 1.88x10 -6 and 6.56x10 -6 M for the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads, respectively. The correlation coefficients (R²) of 0.999 and 0.996 represent the homogeneity of the laccase adsorption process on both tested supports, indicating a monolayer binding. The K d value indicates the strength of the enzyme's binding to the support surface. Consequently, the equilibrium binding constant (K a =1/K d ) derived from the semi-reciprocal plot, and the ∆G values were calculated from the associations ∆G o = −RT ln K a (R is the gas constant (8.314 J mol -1 K -1 ) and T is the temperature in Kelvin) and found to be −32.7 and −29.6 kJ/mol for the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads. The negative ΔG values showed that the immobilization of laccase on the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads was a promising process. Table 1. Langmuir isotherm model constants, correlation coefficients, and ∆G values q e, exp (mg/g) q m (mg/g) K d x10 6 (M) R 2 ∆G (kJ/mol) MCB@PDA-HMDA-Cu(II) 87.9 92.6 5.32 0.999 -32.7 MCB@PDA-HMDA 42.4 49.9 1.53 0.996 -29.6 3.3. Covalent immobilization of laccase on the MCBs@PDA-HMDA-GA beads Laccase was also covalently immobilized on the MCB@PDA-HMDA-GA beads after activation with glutaraldehyde. In this immobilization method, a combination reaction can realize between the amino groups of the matrix and the amine or thiol groups of the enzyme molecules. The grafting of support with PDA-HMDA pairs increased surface area and reactive sites for the covalent immobilization reaction. The maximum laccase immobilization yield of laccase on the MCB@PDA-HMDA-GA beads was 51.6 mg/g, respectively. The recovered activity of the immobilized laccase was about 64.8% on the MCB@PDA-HMDA-GA beads. These reported immobilization efficacy and activity recovery are comparable with earlier studies (Bayramoglu et al., 2012, Kamal et al., 2025. For example, fungal laccase and peroxidase were co-immobilized covalently on a 7% (w/v) polyacrylamide hydrogel and used for biodegradation of BPA, the immobilization efficiency was 78.6 % and activity recovery was 33.7% (Kamal et al., 2025). In these immobilization methods, the measured activity of the adsorbed laccase was significantly lower compared to metal chelate interaction and covalently immobilized laccases. It should be noted that, compared to covalent binding and adsorption methods, the metal chelate interaction method for laccase immobilization showed noticeable advantages in augmenting laccase activity and laccase immobilization capacity (Bayramoglu et al., 2012). This could be due to the presence of Cu(II) on the support, which improved retained enzyme activity. 3.4. Effect of pH and temperature on the activity of laccase preparations The optimal pH values of laccase preparations were investigated in the pH range 3.0–8.0 (Figure S5A). For the free laccase and MCB@PDA-HMDA-Cu(II)-Lac preparations, the optimal activities were detected at around 6.0. Whereas the optimal activity for covalently immobilized enzyme (i.e., MCB@PDA-HMDA-GA-Lac) was observed at pH 6.5. The catalytic activity of the free laccase compared to all immobilized laccase preparations is more pH sensitive, whereas the shape of the curves of the immobilized laccase preparations was significantly broadened (Figure S5A). The observed optimal activity shifts of the covalently immobilized laccase from the optimum pH to a less acidic region could be due to the multipoint covalent attachment of the laccase on the MCB@PDA-HMDA-GA beads. This could increase the conformational rigidity and make it more stable at pH 6.5, thus exhibiting changed catalytic performance (Rybarczyk et al., 2025; Sadeghzadeh et al., 2020; Bayramoglu et al., 2019). Furthermore, the immobilized laccase preparations showed comparatively high activity over a wider pH range compared to the free enzyme. The temperatures activity profiles of the free and immobilized laccase preparations are presented in Figure S5B, the activities of the laccase preparations increased with increasing temperature up to 35 °C, then decreased. However, the immobilized laccase preparations exhibited better temperature stability than their free counterparts (Rodriguez et al., 2025; Bayramoglu et al., 2019). These could result from the formation of relatively stable structures established upon immobilization between enzyme molecules and the functional groups of the support. In particular, the immobilization of the enzyme via multipoint interaction may reduce the conformational flexibility, thus improving its activity space and structure, and may render it more stable at the given temperature (Bayramoglu et al., 2018). Similar observation have been also reported previously, for example, Hydroxyapatite-coated magnetic nanoparticles was used as a support for laccase enzyme immobilization and showed increased stability and catalytic activity in a broader pH range and higher temperatures than its free counterpart, with optimal activity at pH 6.0 (El-Shishtawy et al., 2025). 3.5. Kinetic parameters Kinetic parameters of the enzyme preparations, i.e., K m and V max values, were calculated using syringaldazine as an artificial substrate. The K m values of all the immobilized laccase preparations (i.e., immobilization via adsorption, metal chelate interactions, and covalent binding) were higher than those of the free enzyme (Table 2). The K m values of the immobilized laccase preparations (adsorption, metal chelate interactions, and covalent binding) were increased by about 1.77, 1.33, and 2.55-fold compared to the free laccase. The K m value shows the affinity of the enzyme to its substrate, and a high K m value indicates a low affinity of the enzyme to the substrate. The increase in the K m values of the immobilized laccase preparations could be due to deformation of the active site of the immobilized enzyme preparations, resulting from the reduced mobility of the enzyme's functional groups, which consequently diminishes its affinity to the substrate (Wehaidy et al., 2024). Whereas, the V max value of the immobilized laccase preparations was reduced by about 1.25, 1.07, and 1.45-fold compared to the free enzyme. The declined V max values of all the immobilized laccase preparations could be due to lower accessibility of the immobilized enzymes to interact with the substrate compared to their free counterparts (Bayramoglu et al., 2019). The V max /K m ratio describes the degree of catalytic activity of the enzyme-substrate sets. The catalytic activity (V max /K m ) of the free and immobilized laccase preparations on the MCB@PDA-HMDA-Lac, MCB@PDA-HMDA-Cu(II)-Lac, and MCB@PDA-HMDA-GA-Lac were calculated as 38.8, 60.6, and 23.3, and the catalytic efficiencies of the immobilized preparation were decreased compared to free laccase by about 2.23, 1.43, and 3.72 folds, respectively. The stability of the covalently immobilized laccase is improved due to the multipoint linkage formation, while decreased the flexibility of enzyme for substrate binding. The efficiency factor “η” can be determined from the maximum reaction rates of the immobilized enzymes over that of the free enzyme, and the calculated productivity factors for immobilized laccase preparations are presented in Table 2. ɳ = ѵ i /ѵ f (3) where v i and v f are the reaction rates of the immobilized enzyme and free enzyme, respectively. The decrease in the order of the catalytic efficiency factor of immobilized preparations compared to free laccase was MCB@PDA-HMDA-Cu(II)-Lac > MCB@PDA-HMDA-Lac > MCB@PDA-HMDA-GA-Lac. Table 2. Kinetic parameters of the free and immobilized laccase Enzyme preparations K m (mM) V max (U g -1 ) η V max /K m Free laccase 0.27 23.4 1.0 86.6 MCB@PDA-HMDA-Lac 0.48 18.6 0.79 38.8 MCB@PDA-HMDA-Cu(II)-Lac 0.36 21.8 0.93 60.6 MCB@PDA-HMDA-GA-Lac 0.69 16.1 0.69 23.3 3.6. Reusability, thermal, and storage stabilities of the laccase preparations The reusability performance of the immobilized enzyme is an important factor for large-scale industrial applications (Yu et al., 2025). The covalently immobilized laccase (i.e., MCB@PDA-HMDA-GA-Lac) retained approximately 89.8% of its initial activity after five successive uses (Figure 4A). On the other hand, the MCB@PDA-HMDA-Lac and MCB@PDA-HMDA-Cu(II)-Lac retained about 57.2% and 69.6% of their initial activities, respectively. The excellent reusability of the covalently immobilized laccase may be attributed to the firm fixation of the enzyme to the support, which generates a stable multipoint binding and consequently diminishes the enzyme's release from the support. The long-term storage stability of the laccase preparations was studied by storing them in phosphate buffer solution (20 mmol/L, pH 6.5) at 4 °C for 8 weeks. After this period, the free laccase, MCB@PDA-HMDA-Lac, MCB@PDA-HMDA-Cu(II)-Lac, and MCB@PDA-HMDA-GA-Lac lost 89.2%, 67.5%, 54.8% and 32.4% of their initial activities, respectively Figure 4B. The free laccase retained only 13% its initial activity after 56 days of storage at 4 °C. The stability order of the laccase preparation was MCB@PDA-HMDA-GA-Lac > MCB@PDA-HMDA-Cu(II)-Lac > MCB@PDA-HMDA-Lac > free laccase. The covalent immobilization of the enzyme on the MCB@PDA-HMDA-GA-Lac provided a higher shelf life compared to the other laccase preparations. The improved storage stability of the immobilized laccase preparations can be attributed to the microenvironment of the support materials and the enzyme's fixation via multipoint attachment on these supports, which protects the enzyme from denaturing factors. The thermal stabilities of the laccase preparations were studied in the absence of substrate at two different temperatures (i.e., 55 °C and 70°C). As observed from Figure S6, the immobilized laccase preparations were inactivated at a much slower rate compared to the free enzyme for the studied temperatures. At 55 °C for 120 min incubation time, the MCB@PDA-HMDA-GA-Lac, and MCB@PDA-HMDA-Cu(II)-Lac, maintained their activities approximately 79.3%, and 58.6%, and whereas the free enzyme preserved its activity about 26.4%. At 70 °C for 120 min, the free enzyme lost all its activity after a 60-minute incubation period, while the MCB@PDA-HDMA-GA-Lac and MCB@PDA-HDMA-Cu(II)-Lac preserved about 44.6, and 21.3, of their initial activities, respectively. These results showed that the stabilities of the immobilized laccase preparations to heat were significantly improved upon immobilization. Similar observation have been also reported in the previous studies (Bayramoglu et al., 2019, Borham et al. (2025). Borham et al. (2025) studied the immobilization of fungal laccase onto red seaweed biomass via glutaraldehyde crosslinking, it exhibited high thermal stability, retaining 31% relative activity at 80◦C, while free enzyme totally lost its activity. 3.7. Enzymatic degradation studies 3.7.1. Effect of the presence of mediator on the enzymatic degradation of BPA and RG-5 dye The degradation studies of BPA and RG-5 dye with the free laccase, MCB@PDA-/HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac beads were monitored by taking samples at different time intervals and examined as defined above. As a control, the heat-inactivated immobilized enzyme preparations did not exhibit any degradation activity for BPA and RG-5 dye. However, using active immobilized laccase preparations, substantial changes were detected in the UV–vis absorbance spectra of BPA and RG-5 dye. The degradation rates of BPA and RG-5 dye in the absence and presence of acetosyringone as a mediator chemical with time were studied using MCB@PDA-HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac. The initial concentrations of BPA and RG-5 were 20.0 mg/mL (Figure 5A). As observed from this figure, the degradation rates of both immobilized laccase preparations for BPA and RG-5 dye were significantly enhanced in the presence of acetosyringone compared to their absence. The biodegradation rates of BPA and RG-5 were found to be 98.4% and 78.2% using MCB@PDA-HMDA-Cu(II)-Lac and 83.7% and 67.2% using MCB@PDA-HMDA-GA-Lac, respectively. On the other hand, in the absence of the mediator compound, the degradation performances of MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac were 67.6% and 52.7% for BPA, and 38.3% and 22.8% for RG-5 dye, respectively. As observed from Figure 5A, the degradation performance of the MCB@PDA-HMDA-Cu(II)-Lac preparation for BPA and RG-5 dye was higher for both pollutants compared to MCB@PDA-HMDA-GA-Lac. It should be noted that the free laccase degraded BPA and RG-5 in the presence of the mediator compound, with approximately 37.4% and 13.1% degradation, respectively. In the absence of the mediator, degradation was observed at 21.6% and 9.8%, respectively. These results showed that the immobilized laccase preparations exhibited high performance in degrading both pollutants compared to the free enzyme. These results could be ascribed to enhanced stabilities and increased catalytic performances deliberated by immobilization. It should be noted that the degradation rates of BPA and RG-5 dye in the presence of mediator compound by the MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac were clearly higher than those in the absence of mediator. For all cases, the biodegradation rate of BPA was higher than that of the RG-5 dye for both immobilized laccase preparations, as determined by experiments with a 120-min contact period. Finally, both immobilized laccase preparations exhibited better performance for degradation of BPA and RG-5 dye compared to the free enzyme, and similar observations were stated for the immobilized laccase in the earlier works and many other oxidative enzymes (Bayramoglu et al 2019; Zhank et al., 2022; Zhang et al., 2023; Suhaimi et al., 2025). For examples, Abkenar and Dehnavi (2025) studied the entrapped of laccase enzyme within MIL–100(Fe) metal-organic frameworks. It was used for removal of BPA, and the removal performance of BPA was reported as 83 % from 20 mg/L concentration. Antanaskovic et al studied the immobilization of laccase on biochar via adsorption, and evaluates its application for brilliant green degradation. More than 92% of brilliant green (50 mg/L) was removed within 4 h, at pH 5 and 30°C. 3.7.2. Effect of initial concentration of BPA and RG-5 dye on biodegradation rates The initial concentrations of BPA and RG-5 solutions were varied in the medium between 5.0 and 30.0 mg/L in the presence of the mediator compound. As presented in Figure 5B, the amount of biodegraded BPA and RG-5 by the free enzyme, MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac progressively increased with increasing initial concentrations of the tested pollutants in the medium. When the initial concentrations of BPA and RG-5 dye were higher than 20 mg/L, the degradation rates did not increase meaningfully. At 20 mg/g BPA concentration, the removal rates of BPA with the free laccase, MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac were found to be 37.1%, 96.9%, and 77.8%, respectively, while at 20 mg/g RG-5 concentration, the removal rates were observed as 13.2%, 78.2% and 47.6 %, respectively. As observed in Figure 5B, when the initial concentrations of BPA and RG-5 dye were increased, the likelihood of BPA and/or RG-5 dye molecules interacting with the enzyme in the medium increased, thereby enhancing the removal rates. While the initial concentrations of BPA or RG-5 were further increased, it could become toxic to the enzyme molecules. Thus, this could negatively influence the catalytic activity and degradation performance of the enzyme. Finally, the reduction in the degradation performances of all the laccase preparations at high pollutant concentrations could be due to the poisoning effect of BPA and RG-5 dye and their byproducts. 3.8. Determination of BPA and RG-5 dye degradation rates with MALDI-ToF MS The spectra of the BPA and RG-5 dye achieved at the zero-point, and positive ion MALDI mass spectrum of BPA and RG-5 dye at different time intervals are presented in Figure 6 (partly A and B, respectively). In Figure 6A, the mass spectrum of BPA in the medium showed a monoisotopic peak at 251.1 m/z. After treatment with MCB@PDA-HMDA-Cu(II)-Lac beads, a noticeable decrease in the intensity of the BPA peaks was observed as the contact period increased with the MCB@PDA-HMDA-Cu(II)-Lac preparation. From the signal-to-noise ratios, the percentage removal of BPA was estimated, focusing on the signal-to-noise ratio of the protonated molecular ion peak of the BPA molecule. The percentage of enzymatic degradation of BPA was found to be 47.3%, 76.6%, and 97.7% for 30, 60, and 120 minutes, respectively. At this point, it was observed that the percentage degradation was nearly 97.7% when the contact time was 120 minutes. This elimination proposes that the MCB@PDA-HMDA-Cu(II)-Lac was efficiently degraded by BPA. These results provide valuable insight into the proper degradation of BPA through MCB@PDA-HMDA-Cu(II)-Lac oxidation, suggesting a potential approach for environmental remediation applications. After contact with MCB@PDA-HMDA-Cu(II)-Lac and RG-5 dye for 120 min, the observed new peaks could be attributed to enzymatic degradation products, such as the diazo side chain and –NH-bonding (Figure 6B). From signal/noise ratios, the percentage removal of RG-5 was calculated, focusing on the signal/noise ratio of the protonated molecular ion peak of the dye. The percentage removal of RG-5 (10 mg/L) was 27.3%, 53.6%, and 80.2% at 30, 60, and 120 min, respectively. Here, it was observed that the degradation kinetics of RG-5 dye were significantly slower than those of BPA used in this study. This is the case of the stable phthalocyanine core of RG-5 dye. As reported earlier, laccase is a useful biocatalyst for removing many complex aromatic compounds, such as dyes, pesticides, and chlorinated phenols, from aqueous media (Zhang et al., 2025; Zheng et al., 2023; Vallejo et al., 2025). 3.9. Toxicity studies D. magna is a small planktonic crustacean extensively spread in pools and lakes in many countries, and utilized in many toxicity test studies. D. magna is very susceptible to many toxic chemicals and gives a quick response. The effects of BPA and RG-5 dye concentrations on D. magna mobility were examined for 48 h. Up to 5.0 mg/L concentrations of pure BPA and/or RG-5 dye did not show any toxic effect on D. magna . The EC 50 values of pure BPA and RG-5 dye in 48 h were found to be 13.8 and 7.2 mg/L, respectively. The obtained results are comparable to those of earlier studies, and the reported LC50 values for 48 h ranged from 3.9 to 20.0 mg/L for D. magna (Bayramoglu et al., 2019; Bae and Freeman, 2007). The higher toxicity of RG-5 dye compared to BPA may be attributed to its complex chemical structure. Generally, a longer contact time results in a smaller EC50 value, allowing for the detection of the chemical's toxicity more effectively. At an initial concentration of 25 mg/L, BPA and RG-5 dye were treated with the MCB@PDA-HMDA-Cu(II)-Lac preparation for 120 min, and the toxicity of the samples was evaluated using D. magna . After treatment of BPA and RG-5 for 120 min, the enzymatically treated solution of BPA did not show any toxic effect on D. magna , while the byproducts of RG-5 dye displayed a toxic effect on D. magna . The immobility of D. magna was about 48%. Freshwater algae species are frequently used as bio-indicators for toxicity testing of many chemicals. Among them, Chlorella vulgaris is highly sensitive to numerous chemicals and is commonly used in toxicity studies. The growth inhibition test of BPA and RG-5 dye was performed on C. vulgaris at three different concentrations of BPA and/or RG-5 dye (i.e., 5.0, 10, and 25.0 mg/L). The degradation byproducts of the BPA and RG-5 dye were obtained after treatment with the MCB@PDA-HMDA-Cu(II)-Lac preparation for 120 min. The initial concentration of each pollutant was 25 mg/g. These byproducts were also used in the algal growth inhibition test as described above. As shown in Table S1, the reduction in the growth rate of C. vulgaris for BPA and RG-5 dye was found to be dependent on the concentration of these test compounds. An increase in the initial BPA or RG-5 concentration resulted in a reduction in the harvested biomass weight. The maximum harvested biomass of C. vulgaris in the control culture was 0.817 g/L dry weight, achieved after seven days of incubation. At a 25 mg/g BPA concentration, the harvested biomass of C. vulgaris was considerably reduced and found to be 0.358 g/L dry biomass after a seven-day incubation period. The C. vulgaris grew at low concentrations of BPA and RG-5 dye at 5.0 mg/L. On the other hand, at a concentration of 25 mg/g of RG-5, the amount of biomass harvested decreased significantly to 0.173 mg/g (Table S1), compared to the control (0.817 g/L). According to these observations, experiments conducted with C. vulgaris and RG-5 dye showed more toxic effects than those of BPA. It should be noted that numerous reports have been published on the toxicity assessments of dyes using various freshwater alga species; however, no report is currently available on the toxicity assessments of RG-5 dye using alga species. After 120 min of enzymatic treatment of BPA and RG-5, there was no inhibition of the growth of algae, while the treated RG-5 sample showed 77% growth inhibition. Therefore, the presented work may provide important information to define the potential risk of RG-5 dye toxicity on the freshwater algal species. Conclusion In the present study, magnetic cellulose beads MCB were prepared via the extrusion-dropping method and then grafted with PDA-HMDA. The laccase was immobilized via copper-ion coordination or covalently on the MCB@PDA-HMDA beads after Cu(II) chelating or GA activation, respectively. In comparison with MCB@PDA-HMDA-Cu(II)-Lac preparation, the covalently immobilized laccase (i.e., MCB@PDA-HMDA-GA-Lac) displayed high stability and good reusability performance after five runs. On the other hand, the immobilized laccase via metal chelate interaction exhibited high recovered activity and effectively degraded BPA and RG-5 dye compared to the covalently immobilized counterpart. The degradation of BPA and RG-5 was achieved using both free and immobilized laccase preparations in a batch system. The degradation percentage of BPA (20 mg/L) and RG-5 dye (10 mg/L) was obtained after contact with MCB@PDA-HMDA-Cu(II)-Lac in the presence of mediator compounds for 120 min, and up to 98.4% degradation of BPA was observed, whereas RG-5 was degraded about 78.2% after 120 min incubation. While MCBs@PDA-HMDA-GA-Lac degraded about 83.7 and 67.2 % of BPA and RG-5, respectively. The MCB@PDA-HMDA-Cu(II)-Lac preparation showed good degradation performance for both tested pollutants. Biodegradation rates of BPA and RG-5 were also determined using MALDI-TOF-MS spectra. This study also investigated the toxicological properties of BPA and RG-5 dye, providing an understanding of the toxicity risk of BPA and RG-5 on the mobility of D. magna and C. vulgaris . Declarations Author Contribution Gulay Bayramoglu: Methodology, Investigation, Validation, Formal analysis, Resources, Writing – original draft, Writing – review & editing. M. Yakup Arica: Conceptualization, Writing - review & editing. 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2","display":"","copyAsset":false,"role":"figure","size":1169056,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image of the magnetic nanoparticles (A), light microscope images of the pure cellulose beads and MCB@PDA-HMDA beads (B), and magnetic separation of MCB@PDA-HMDA beads from solution (C).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/d5ede69b7430cb3558ca73cf.png"},{"id":99811294,"identity":"5da2644d-0bf7-447f-a324-6541f3b1fd90","added_by":"auto","created_at":"2026-01-08 14:33:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32421,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of enzyme concentration on immobilization efficiency of MCB@PDA-HMDA, MCB@PDA-HMDA-Cu(II)-Lac \u003cstrong\u003e(A),\u003c/strong\u003e Effect of pH on immobilization efficiency of MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II)-Lac \u003cstrong\u003e(B)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/6f39bb0b9811c0281c065eca.png"},{"id":99811539,"identity":"4b98a40b-7107-4355-8f84-6113a1e3910c","added_by":"auto","created_at":"2026-01-08 14:34:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47285,"visible":true,"origin":"","legend":"\u003cp\u003eReuse numbers of\u003cstrong\u003e \u003c/strong\u003eMCB@PDA-HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac \u003cstrong\u003e(A),\u003c/strong\u003e Storage stabilities of free, MCB@PDA-HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac preparations (B)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/3f6984847fe6225280ad723b.png"},{"id":99811350,"identity":"03e3bbfc-10b5-4add-b23d-a18000a7dc2d","added_by":"auto","created_at":"2026-01-08 14:33:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61839,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation performance of\u003cstrong\u003e \u003c/strong\u003eMCB@PDA-HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac preparations for BPA and RG-5 dye in the absence (MA) and presence (MP) of mediator compounds \u003cstrong\u003e(A)\u003c/strong\u003e, Effect of initial concentration of BPA and RG-5 dye on the degradation rates \u003cstrong\u003e(B)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/321811fec29e1e882bab44a9.png"},{"id":99811067,"identity":"247a6cb6-2c59-4c1d-a909-c4b58f698136","added_by":"auto","created_at":"2026-01-08 14:33:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":100364,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation of BPA (20 mg/L) using MCB@PDA-HMDA-Cu(II)-Lac preparation in the presence of mediator compounds. High resolution MALDI mass spectra representing intensity fading of the monoisotopic signal of BPA [M+Na]\u003csup\u003e+\u003c/sup\u003e at the beginning of time zero. MALDI-TOF mass spectra are based on the relative intensity of the y-axis of depicted peaks (the highest peak was accepted as 100%). (A) 0 min. (B) 30 min. (C) 60 min. (D) 120 min \u003cstrong\u003e(A), \u003c/strong\u003eDegradation of RG-5 using MCB@PDA-HMDA-Cu(II)-Lac preparation in the presence of mediator compounds (A) 0 min. (B) 30 min. (C) 60 min. (D) 120 min \u003cstrong\u003e(B).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/82f2749a8622c0a7897c207d.png"},{"id":99814847,"identity":"026f4d72-9e03-4df5-8d08-3db91d3a6d7a","added_by":"auto","created_at":"2026-01-08 14:42:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2708742,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/c894f28b-e903-4161-9970-1f73a7bcce26.pdf"},{"id":99811460,"identity":"7efb73a4-9bb7-4a07-81d5-6b786ef2a052","added_by":"auto","created_at":"2026-01-08 14:34:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1640321,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8013262/v1/d8f5f0e8f6b530072ab0f71d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immobilization of laccase on magnetic cellulose beads for enhanced biodegradation of bisphenol A and a metal-complex dye","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe biodegradation of pollutants from wastewaters using industrially important enzymes can be achieved through a few reaction steps under mild reaction conditions, resulting in reduced waste. Considering the important roles of enzymatic reactions, the high cost, low stability, and limited reuse numbers of free enzymes limit their extensive applications in biotechnological areas (Antanaskovic et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Arica et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bayramoglu and Arica, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Borges et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The fixation of enzymes on insoluble supports can significantly reduce the limitations mentioned above by enhancing their operational stability under various experimental conditions, thereby dominating the enzymatic reaction and permitting consecutive operations (Abkenar and Dehnavi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bayramoglu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Borham et al.,2025; Garg et al ., 2024; Hoang and Kim, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Maine et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The choice of support and method for enzyme immobilization is crucial for preserving the activity of immobilized enzymes for biotechnological applications. Various organic and inorganic support materials, including cellulose, chitosan, poly(acrylic) polymers, polymeric resins, clay minerals, magnetic particles, and silica nanoparticles, have been utilized for the immobilization of enzymes (Hussain et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ishak et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bayramoglu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; El-Shishtawy et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Gurgel et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Most of the enzymes on these support materials have been immobilized via chemical and physical methods. The former is the most widely employed method, which generates a covalent linkage between enzymes and the support (Alokpa et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., Lu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Souza et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This method provides many advantages, such as minimal enzyme leakage from support, a high amount of enzyme loading, repeated usability, and improving thermal and pH stabilities of enzymes compared to the physical immobilization method (Moayedi and Yousefi, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Mota and Gimenez, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Oraby et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Othman and Flaifil, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Particularly, supports materials with pendant amine groups can be activated with various bifunctional agents, such as glutaraldehyde and carbodiimide, to react and form a covalent linkage with different groups on the surface of the enzyme molecules, including amino, hydroxyl, and thiol groups. These linkages can provide negligible deformation to the three-dimensional structure with minimal chemical influence on the structure of enzymes (Bayramoglu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Feng et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, glutaraldehyde-activated supports can be practically ideal for the covalent immobilization of enzymes on both laboratory and industrial scales (Bayramoglu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Glutaraldehyde-activated supports can react with enzymes under mild reaction conditions and over a wider pH range, allowing for facile covalent interactions. Moreover, the bond formed between the enzyme and the supports is very stable under wet storage conditions. Therefore, glutaraldehyde-activated supports have been widely used for the immobilization of proteins, such as glutaraldehyde-activated acrylic polymers, polyethyleneimine-modified various microbeads (Bayramoglu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khanam et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), microspheres containing amine groups, and magnetic nanoparticles, which have been utilized in earlier enzyme immobilization studies.\u003c/p\u003e \u003cp\u003eCellulose beads can be prepared with diameters ranging from micro to millimeters and used in a variety of complex applications, such as chromatographic fields, enzyme immobilization technology, and drug delivery systems. Cellulose and its derivatives possess several essential properties that make them suitable for the applications above, including sustainability, non-toxicity, biocompatibility, biodegradability, and good mechanical properties (Bayramoglu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sourgi and Dehnavi, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cellulose is made of D-glucopyranosyl units and linked with each other via β-1.4-glycosidic bonds, and each sugar unit has three available hydroxyl groups on C2, C3, and C6 (Bao et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Oktaviani et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Udoetoka et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Particularly, cellulose-based materials have been highly recommended as an ideal carrier for enzyme immobilization (Xing et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yan and Hou, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cellulose-based materials have garnered important attention as enzyme immobilization materials due to their distinctive structural properties, which include an interior porous assembly, a large surface area, and high numbers hydroxyl groups that are easily chemically variable (Lin et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The hydroxyl groups on cellulose fibers have not formed easily covalent bonds with enzymes; chemical alteration is necessary to present useful groups, such as amino groups, to simplify covalent immobilization. Enzymes can be covalently immobilized on cellulose fibers to enhance enzymatic efficiency and production yields, while also contributing to green technology and sustainable sources. Many enzymes have been immobilized on cellulose-based materials, including laccase, trypsin, lipase, glucose oxidase, peroxidase, and tyrosinase (Kim et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mota and Gimenez, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sathishkumara et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), among others. Moreover, cellulose-based magnetic supports can permit easy stop of the reaction using an external magnet, and also provide easy reusability.\u003c/p\u003e \u003cp\u003eLaccase EC 1.10.3.2) is an oxidation\u0026ndash;reduction enzyme and produced by many plants and fungi, and containing a copper atom in its catalytic center. Laccase is capable of degrading numerous phenolic compounds, including various dyes, phenols, and complex aromatic compounds. The critical limitation of the use of laccase in pollutant biodegradation is low stability and short persistence of the free enzyme, as well as the high production cost of the enzyme (Aghera et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Al- Sareji et al., 2023; Bayramoglu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Petrila et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These important problems can be eliminated by immobilization, thereby increasing their economic value (De Paula et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Jin et al., 2025; Kamal et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). An efficient immobilization approach for laccase relies on the constitution and performance of the support, as well as the choice of immobilization method. Among numerous immobilization protocols, covalent coupling is a desired method, as it allows the enzyme to be tightly bound to the support (Kokar et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kumar and Sridhar, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kyomuhimbo and Brink, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this work, laccase from a white rot fungus (\u003cem\u003eTrametes versicolor\u003c/em\u003e) was immobilized on the PDA-HMDA grafted magnetic cellulose beads (i.e., MCB@PDA-HDMA) either through metal chelate interaction or glutaraldehyde coupling. For this, the magnetic cellulose beads were synthesized and coated with PDA in the presence of HMDA. In the second step, for immobilization via metal chelate interaction, Cu(II) ions were chelated onto the beads, or the beads were activated with GA for covalent attachment. Characterization of the immobilized enzyme preparations was investigated under different experiments using free laccase as a control system. The optimum pH and temperature, kinetic parameters, stability, and reusability of both free and immobilized enzymes were studied. The immobilized enzyme preparations were used to degrade BPA and a metal dye, RG-5, from an aqueous medium. Results showed that the performance of the immobilized laccase preparations in degrading BPA and RG-5 dye was very effective; hence, the presented immobilized methods could provide a new approach to removing phenolic compounds from industrial effluents. To the best of our knowledge, the presented laccase immobilized methods and RG-5 degradation have not been reported. Also, the support materials as prepared were not used for any enzyme immobilization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLaccase (EC 1.10.3.2, 20 U/mg solid) from \u003cem\u003eTrametes versicolor,\u0026nbsp;\u003c/em\u003e4-hydroxy-3,5-dimethoxyhydroxybenzaldehyde (syringaldazine), microcrystalline cellulose (20\u0026ndash;160 \u0026micro;m), dopamine, Tris-HCl, hexamethylenediamine, glutaraldehyde solution (25 %), Bisphenol A, Reactive Green 5 \u0026nbsp; (Procion Green H 4G), and sodium hydroxide were supplied from Sigma-Aldrich Chem. Co, Germany.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e particles were synthesized via thermal precipitation of Fe(III) and Fe(II) as reported earlier (Arica et al., 2017). \u0026nbsp;Briefly, 200\u0026thinsp;mL of FeCl\u003csub\u003e3\u003c/sub\u003e (0.3\u0026thinsp;mol/L) and 100\u0026thinsp;mL of ethylene glycol were added to a reaction vessel and magnetically agitated for approximately 10\u0026thinsp;min. Then, 200 mL of FeCl\u003csub\u003e2\u003c/sub\u003e (0.1 mol/L) and polyethylene glycol (PEG, 6000; 2.0%) mixture was transferred into the reaction medium and agitated for an additional 30 min. After this period, 50 mL of ammonia solution (25%, v/v) was added to the reaction vessel and refluxed at 70 \u0026deg;C for 2.0 h, followed by an additional 1.0 h at 90 \u0026deg;C. The magnetic particles were dried under reduced pressure at 25 \u0026deg;C. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Preparation of MSB and grafting with PDA-HMDA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCB were prepared via the extrusion-dropping method as described in an earlier work (Bayramoglu et al., 2024). An alkaline solution was prepared by adding 14 g of NaOH and 24 g of urea to 162 mL of H₂O and stirring the mixture magnetically at 15 \u0026ordm;C for 30 min. Then, 6.0 g of cellulose microcrystalline powder and 4.0 g of magnetic nanoparticles were transferred to the medium, and further stirred at 250 rpm for 60 min. The resulting blend was dropped into the sodium chloride solution (400 mL, 15%) using a syringe pump. The prepared MCB was collected using an external magnet and cleaned with deionized water.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dopamine molecule has catechol and amine functional groups, and under alkaline conditions, dopamine self-polymerizes to form PDA and generates coating layers on the surface of different organic and/or inorganic materials. PDA has several distinctive possessions, including biocompatibility, strong adhesion, and antioxidant properties. For PDA/HMDA coating, the magnetic cellulose beads (2.0 g) were transferred to a reaction vessel containing 99 mL of Tris-HCl buffer (pH 8.5). Then, 600 mg of dopamine and 1.0 mL of HMDA were added, and stirred magnetically for 10 min. After that, the reaction vessel was placed on an orbital shaker and the grafting reaction was performed at 50 \u0026ordm;C for 18.0 h. After the reaction period, the MCB@PDA-HMDA beads were cleaned with distilled water to remove any unreacted dopamine and HMDA. Next, the MCB@PDA-HMDA (2.0 g) was transferred to a glutaraldehyde (GA) solution (100 mL, 1.0%) to produce functionalized magnetic cellulose beads (MCB@PDA-HMDA-GA). The activation reaction was carried out at 50 \u0026ordm;C while continuously agitating for 4.0 h. The MCB@PDA-GA were then washed with an acetic acid solution (1.0%, v/v) and subsequently with deionized water to remove any reaction impurities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSome of the MCB@PDA-HMDA beads were chelated with Cu(II) ions for immobilization of laccase via metal-chelate interaction. For this, Cu(II) solution (100 ppm) was prepared from CuCl\u003csub\u003e2\u003c/sub\u003e in deionized water at a pH of 4.5. The MCB@PDA-HMDA beads (4.0 g) were transferred to the Cu(II) solution (100 mL) and stirred at 100 rpm and at 25 \u0026deg;C for 2.0 h. Then, the Cu(II) ions chelated MCB@PDA-HMDA beads were washed with phosphate buffer solution (50 mM, pH 6.5). The initial and final concentrations of Cu(II) ions in the medium were determined using a flame atomic absorption spectrophotometer (AAS, Shimadzu AA6800, Japan). At least 10 AAS measurements were recorded, and the mean of the data was used. The amount of chelated Cu(II) ions (\u0026mu;mol/g beads) was determined as the change in the before and after contacted he beads Cu(II) ions solution. The leakage of the chelated Cu(II) ions from the beads was examined in the pH range of 5.0\u0026ndash;9.0. The MCB@PDA-HMDA-Cu(II) beads were incubated with Cu(II) ion solution at different pH values while stirring at 25 \u0026ordm;C for 12 h, and the released Cu(II) ion concentration was detected in the supernatants using AAS as described above. Subsequently, MCB@PDA-HMDA-GA and MCB@PDA-HMDA-Cu(II) beads were utilized for laccase immobilization, with MCB@PDA-HMDA beads serving as a control system. The preparation step of support materials is schematically presented in Figure 1.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Immobilization of laccase via adsorption and metal chelate interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immobilization of laccase on MCB@PDA-HMDA-Cu(II) was studied based on the incorporation of borderline Cu(II) ions on the chelating groups of MCB@PDA-HMDA functionality, and also MCB@PDA-HMDA was used as a control system. Immobilization of laccase on the MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II) beads was realized at different pH values, in either acetate (100 mmol/L, pH 4.0\u0026ndash;5.0) or in phosphate buffer (100 mmol/L, pH 6.0\u0026ndash;9.0). The initial laccase concentration was 2.0 mg/mL in the corresponding buffer solution, and immobilization of laccase via adsorption and metal chelate interaction was performed at 17 \u0026deg;C while stirring for 2.0 h. Then, the laccase immobilized on both magnetic beads was collected magnetically from the medium, and cleaned with the corresponding buffer solution. The amount of protein in the solutions was determined by the Bradford method (1976) as described previously.\u003c/p\u003e\n\u003cp\u003eTo determine the reusability of MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II) beads, the laccase adsorption/desorption round was repeated five times by using the same support materials. The laccase desorption from the MCB@PDA-HMDA and MCB@PDA-HMDA-Cu(II) beads was achieved using a KSCN solution as the desorption agent (10 mL, 1.0 mol/L, pH 8.0). The laccase-laden MCB@PDA-HMDA or MCB@PDA-HMDA-Cu(II) beads were added in the desorption medium and stirred magnetically at 150 rpm and at room temperature for 2.0 h. The samples were cleaned, then reutilized in the next run for immobilization of laccase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Immobilization of laccase via covalent attachment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe glutaraldehyde-activated beads (i.e., MCB@PDA-HMDA-GA, 1.0 g) were transferred to 2.0 mg/mL of laccase solution (10.0 mL, pH 8.0, 15 \u0026ordm;C) and mixed for 4.0 h in a rotated incubator shaker. Then, the laccase-immobilized beads (i.e., MCBs@PDA-HMDA-Lac) were cleaned with phosphate buffer solution (50 mM, pH 6.5). The amount of protein in the solutions was determined at 595 nm using a UV/Vis spectrophotometer (PG Instruments Ltd., Model T80 +, PRC). A BSA standard curve was used to calculate the protein amount in the solutions.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Activity assays of laccase preparations and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ekinetic parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe laccase activity assays were studied over the pH range of 3.0\u0026ndash;8.0 at 25 \u0026deg;C, as well as at temperatures between 15 and 60 \u0026deg;C and at pH 6.0. In these experiments, the laccase activity was measured the oxidation rate of syringaldazine at 530 nm as reported earlier (Bayramoglu et al., 2019). The relative activities of the laccase preparations were considered as the ratio between the measured activities at the tested pH or temperature and the highest level of activity. The K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e values of the laccase preparations were determined using different concentrations (0.1-2.0 mmol/L) of syringaldazine as substrate in acetate buffer at pH 6.0 and at 25\u0026deg;C. The kinetic parameters of the enzyme preparations were calculated using the Michaelis-Menten equations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Thermal and storage stabilities of the laccase preparations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thermal and storage stabilities of the enzyme preparations were evaluated as reported previously (Bayramoglu et al., 2019). The thermal stabilities of laccase preparations were detected after incubation at various temperatures (15\u0026ndash;65 \u0026ordm;C) in phosphate buffer at pH 6.0 for 120 minutes. The storage stability of the laccase preparations was measured at one-week intervals over an 8-week period while storing at 4 ◦C, and the initial activity was defined as 100%. The reusability of laccase preparations was determined by measuring the activity of the same sample five times, and each experiment was triplicated.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Degradation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBisphenol A and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eReactive Green 5 with laccase preparations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reaction mixture containing BPA and RG-5 dye (each 50 mg/L) in phosphate buffer solution (pH 6.0, 50 mM, 5.0 mL) or adding acetosyringone (0.2 mM) in the same buffer. The laccase preparations in BPA or RG-5 solutions were incubated on an orbital shaker at 25 \u0026deg;C and 100 rpm for 120 min. Then, the reduction in the absorbance value of the BPA and RG-5 dye-containing medium was measured at 276 and 670 nm, respectively, using a UV/visible spectrophotometer.\u003c/p\u003e\n\u003cp\u003eThe percentage of the removed pollutant was calculated as:\u003c/p\u003e\n\u003cp\u003eRemoval pollutant (%) = [(\u003cem\u003eC\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e \u0026minus; \u003cem\u003eC\u003c/em\u003e)\u003cem\u003e/C\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e]\u0026nbsp;\u0026times;\u0026nbsp;100 (1)\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e and \u003cem\u003eC\u0026nbsp;\u003c/em\u003eare the concentration of BPA or RG-5 dye in the initial medium and after time t (mg/L). After a given time period, a sample was removed and measured spectrophotometrically. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9. MALDI-ToF- MS studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MALDI-ToF-MS analysis was performed as described previously (Bayramoglu et al., 2019). The mass spectra were achieved on a MALDI-ToF mass spectrometer (Voyager-DE\u0026trade; PRO, Applied Biosystem, USA) at 337\u0026thinsp;nm. The spectra were obtained in positive-ion and linear mode, with an average of 100 shots.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. Eco-toxicity studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChlorella vulgaris\u003c/em\u003e was used in the algal growth inhibition tests according to the OECD guideline 201 (O.E.C.D. 2011). The growth rates of \u003cem\u003eC. vulgaris\u003c/em\u003e were obtained at different BPA and/or RG-5 dye concentrations in the range of 5.0-25 mg/L for a 5-day period. The same test was also performed after 120 min of enzymatic treatment of the BPA and RG-5 dye solutions. The initial algal cell density of the medium was approximately 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL, and cell counts were measured spectrophotometrically at 685 nm. Control groups were also included in the absence of BPA and RG-5, and were used for evaluating the results of ecotoxicological experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe immobilization test of \u003cem\u003eDaphnia magna\u003c/em\u003e was also conducted using neonates (less than 24 hours old) in accordance with the guidelines of OECD No. 202 (OECD, 2004). The concentrations of BPA or RG-5 were 2, 5, 10, 15, 20, and 25 mg/L. Briefly, ten daphnids were transferred to 25 mL medium containing various concentrations of BPA or RG-5. The immobilization rates were calculated after 48 h of exposure. Each treatment test was triplicated.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. Characterization of modified cellulose-based supports\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe available surface amine groups of the MCB@PDA-HMDA beads were determined with a potentiometric titration method. For this, 0.5 g of MCB@PDA-HMDA beads were added to the HCl solution (0.1 mol/L, 10 mL) and incubated in a rotary shaker at 25 \u0026ordm;C for 4.0 h. Then, the final concentration of HCl in the medium was measured using NaOH solution (0.05 mol/L). The specific surface areas of MCB and MCB@PDA-HMDA were determined by the BET method (Brunauer, Emmett, and Teller) using BET Surface Area Analyzers (Quantachrome Nova 2200 E, USA). Former to analysis, samples were incubated at 110 \u0026deg;C for 18 h under reduced pressure. The specific surface areas of the samples were determined. The FTIR spectra of the MNP, MCB, MCB@PDA-HMDA, and MCB@PDA-HMDA-Lac beads were obtained using a Nicolet TM ISTM 50 FTIR spectrometer (Thermo Fisher Scientific, USA). The surface morphologies of the MCB and MCB@PDA-HMDA beads were obtained using a scanning electron microscope (JEOL, Model JSM-5600, Tokyo, Japan). The transmission electron micrograph (TEM) images of the magnetic nanoparticles (MNP) were attained at an accelerating voltage of 120 kV using a JEM-1400 Plus electron microscope in TEM mode (JEOL Ltd, Akishima, Tokyo, Japan). The magnetic properties of the MNP, MCB, MCB@PDA-HMDA, and MCBs@PDA-HMDA were studied at room temperature using a vibratory sampling magnetometer (VSM; Model 155, Digital Measurement System Inc., Westwood, MA, USA). The X-ray diffraction (XRD) patterns of the MNP and MCBs@PDA-HMDA-Lac beads were obtained using Cu-K\u0026alpha; radiation, between 2\u0026theta; of 20 and 80\u0026ordm;, with a 0.1 increment and 2\u0026ordm; min\u003csup\u003e\u0026minus;1\u003c/sup\u003e scan speed (MiniFlex 600, Rigaku).\u0026nbsp;\u003c/p\u003e"},{"header":"Results and Discussions","content":"\u003cp\u003e\u003cstrong\u003e3.1. Characterization of the as-prepared cellulose‑based support materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino group content of MCB@PDA-HMDA beads was determined to be 1.28 mmol/g. The specific surface areas of \u0026nbsp;MCB and MCB@PDA-HMDA beads were determined as 27.8 and 19.7 m\u003csup\u003e2\u003c/sup\u003e/g, respectively. The results exhibited that the specific surface area decreased after grafting of MCB with PDA-HMDA polymers. The resduce in the surface area of the MCB beads after grafting with PDA-HMDA can result from the lessening in the pore sizes and pore volume upon integration of PDA-HMDA polymer molecules on the surface of the MCB beads.\u003c/p\u003e\n\u003cp\u003eThe ATR-FTIR spectra of pure cellulose, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, MCB, MCB@PDA-HMDA, MCB@PDA-HMDA-Lac, and @PDA-HMDA-Cu(II)-Lac are displayed in Figure S1. For the pure cellulose, at 3338 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, an extensive band was observed for the stretching vibration of -OH groups of cellulose (Figure S1A). Two peaks at 2892 and 1640 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e were seen due to stretching and vibration of C-H and C\u0026ndash;C of pure cellulose, respectively. As seen in Figure S1B, a strong peak for the Fe-O bond was observed at 558 cm\u003csup\u003e-1\u003c/sup\u003e, whereas the peaks at 1628 cm\u003csup\u003e-1\u003c/sup\u003e and 3285 cm\u003csup\u003e-1\u003c/sup\u003e could be characteristic of hydroxyl groups associated with H\u003csub\u003e2\u003c/sub\u003eO existence on the surface of the micro particles. The FTIR spectrum of MCB was similar to that of pure cellulose except that a wide adsorption band at 3452 cm\u003csup\u003e-1\u003c/sup\u003e was observed (Figure S1C). This band was related to the stretching vibration of the \u0026ndash;OH groups of cellulose and Fe-OH. The MCB@PDA-HMDA material was also examined using ATR-FTIR to confirm the PDA-HMDA grafted on the surfaces of MCB. As given in Figure S1D, the FTIR spectrum of the PDA-HMDA grafted beads displaying a comparatively extensive band in the 3305 cm\u003csup\u003e-1\u003c/sup\u003e could be attributed to the stretching vibration of hydroxyl groups of cellulose/Fe-OH, and also amine groups of chitosan/HMDA. The observed peak at 1054 cm\u003csup\u003e-1\u003c/sup\u003e could be due to the \u0026ndash;NH shearing vibration of the amide groups. This observation could be attributed to the hydrogen bonds between the PDA and HMDA molecules. These observations showed that PDA-HMDA effectively grafted onto the MCB. All these detected variations were associated with the grafting of PDA-HMDA on the MCB beads.\u003c/p\u003e\n\u003cp\u003eThe FTIR spectra obtained after the immobilization of laccase showed significant changes in the vibration bands in the 1050\u0026ndash;1650 cm\u003csup\u003e-1\u003c/sup\u003e region, indicating that the enzyme was immobilized on the MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-Lac beads (Figure S1E and S1F, respectively). The observed peaks from the FTIR spectrum of the MCB@PDA-HMDA-Cu(II)-Lac beads at 1552 cm\u003csup\u003e-1\u003c/sup\u003e and 1411 cm\u003csup\u003e-1\u003c/sup\u003e originated from the immobilized laccase. Furthermore, the peak at 1634 cm\u003csup\u003e-1\u003c/sup\u003e could be due to the stretching vibration of carbonyl (\u0026ndash;C=O) groups. The peaks were observed at 1552 cm\u003csup\u003e-1\u003c/sup\u003e and between 2897 cm\u003csup\u003e-1\u003c/sup\u003e and 3326 cm\u003csup\u003e-1\u003c/sup\u003e, which could be attributed to the N\u0026ndash;H bending vibration (Figure S1E). Additionally, the peak at 3446 cm\u003csup\u003e-1\u003c/sup\u003e was raised to the \u0026ndash;NH stretching at resonance. The observation of these characteristic peaks on the MCB@PDA-HMDA-Cu(II)-Lac beads corroborates the successful immobilization of laccase via adsorption. As observed in Figure S1F, the reduction in peak extent at 3320 cm\u003csup\u003e-1\u003c/sup\u003e indicates that laccase was successfully immobilized on MCB@PDA-HMDA-Lac. These results also demonstrated that laccase was effectively immobilized on the MCB@PDA-HMDA beads (Figure S1F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure S2 shows SEM images of the dry MCB@PDA-HMDA beads at two different magnifications. As seen in this figure, the MCB@PDA-HMDA beads were spherical in geometry and had an irregular surface appearance (Figure S2A). This could be related to the presence of MNP in the bead formulation. The presence of rough surfaces on the beads can provide high enzyme immobilization capacity, and also increase the surface area for the enzymatic reaction (Figure S2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe TEM nanograph of the Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e nanoparticles in the dry state is presented in Figure 2A. As shown in the figure, the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles had a nearly spherical shape with a diameter of approximately 12 nm. The photographs of the pure cellulose and MNP-entrapped cellulose beads are presented in Figure 2B. The pure cellulose beads were white in color, whereas the MCB@PDA-HMDA beads were black due to the existence of MNP, signifying that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles were uniformly entrapped in the cellulose beads. The average size of the PDA-HMDA grafted cellulose beads was around 1.2\u0026plusmn;0.13 mm (Figure 2B). The MCB@PDA-HMDA beads were simply removed from the reaction medium using an external magnet, demonstrating the sensitivity of the as-prepared magnetic beads to the applied magnetic force (Figure 2C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe magnetization data of the samples are presented in Figure S3. The saturation magnetization of the as-prepared samples was found to be 56.4, 32.6, and 29.3 emu/g for the MCB@PDA-HMDA-Lac beads, respectively. It was observed that saturation magnetization highly relies on the total amount of the MNP in the beads and directly influences the magnetic properties of the materials. As shown in this figure, the magnetization curves of the beads exhibited zero refraction tendency, indicating superparamagnetic properties. The presented data exhibited that the MCB@PDA-HMDA-Lac beads can be simply collected from the solution using an external magnet. The superparamagnetic nanoparticles incorporated into the beads were well characterized, and the targeted success was achieved; the MCB@PDA-HMDA-Lac beads did not clump together due to interactions with each other. Furthermore, the prepared magnetic beads were very stable, and the magnetic properties of the as-prepared support were sufficient. Thus, the support material was easily removed magnetically from the reaction medium within a short time and could be simply re-dispersed within a few seconds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn XRD analyses, the diffraction peaks of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles within the MCB@PDA-HMDA-GA-Lac beads were completely observed. These data indicate that the construction of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e was wholly well-preserved in the MCB@PDA-HMDA-GA-Lac beads during the preparation reactions (Figure S4). As shown in this figure, the MCB@PDA-HMDA-GA-Lac beads exhibit the similar diffraction peaks as the MNP, demonstrating that the magnetic properties were well-preserved. These data indicate that the magnetic properties of the MNP remained unchanged during the preparation step of the support. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Immobilization of laccase on the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMCB@PDA-HMDA-Cu(II) via metal-chelate interaction and MCB@PDA-HMDA beads via adsorption\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of immobilized laccase via metal chelate interaction with Cu(II) ions and adsorption was determined using the Bradford method (1976). The influence of the initial enzyme concentration on the adsorption performance of the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads was studied at pH 6.0 and 25 \u0026ordm;C for 2.0 h, and the enzyme concentrations were varied in the range 0.1-3.0 mg/mL in the medium. The amount of adsorbed laccase augmented with increasing initial concentration of the enzyme in the medium. The maximum immobilization capacities were found to be 87.9 and 42.4 mg/g for MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads, and the amounts of immobilized laccase per unit mass of the both support reached a plateau value at 2.0 mg enzyme/mL, and the retained laccase activity were found to be 81.6 %, and 48.7 comparable with the related literature, respectively (Bayramoglu et al., 2019). As shown in Figure 3A, the lowest amount of laccase was immobilized on the MCB@PDA-HMDA beads via the adsorption method compared to the metal chelate interaction on the MCB@PDA-HMDA-Cu(II). This could be resulted from the coordination complex of Cu(II) ions with the amino acid residues on the surface of the enzyme. The most important amino acid residues on the surface of laccase enzymes are the imidazole group of histidine, thiol group of cysteine, amino groups of lysine and arginine, and carboxyl groups of aspartic acid and glutamic acid, and these residues can make metal chelate interactions with Cu(II) ions. As reported earlier, an appropriate amount of Cu(II) ions preserves the three-dimensional active conformational structure of laccase, allowing it to bind to the substrate and undergo oxidation, thereby enhancing its activity (Bayramoglu et al., 2012; Jing et al., 2025; Li et al., 2024). Immobilization of laccase via Cu(II) ions interaction onto the surface of the MCB@PDA-HMDA-Cu(II) beads\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehas enhanced the immobilization efficiency and enzyme activity compared to immobilization via adsorption (i.e., MCB@PDA-HMDA-Lac)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eto a certain level (Bayramoglu et al., 2012; Plesner et al., 2025; Ranimol and Sunkar 2022). The activity of the MCB@PDA-HMDA-Cu(II)-Lac was remarkably higher than that of the MCB@PDA-HMDA-Lac preparation. This result could be due to the presence of Cu(II) ions on the support, which affected the laccase activity. Because laccase enzyme is a multi-copper oxidase enzyme and needs copper ions to preserve its activity (Plesner et al., 2025). As reported in earlier studies (Bayramoglu et al., 2012), laccases require copper ions to maintain their active conformational structure, bind to their substrate, and facilitate electron transfer. Therefore, immobilization of laccase with Cu(II) chelate formation was also considered in this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe influence of pH on the laccase immobilization efficacy and performance on the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads was studied in the pH range of 4.0-9.0 at 25 \u0026ordm;C with an initial laccase concentration 1.0 mg/mL. The maximum amount of enzyme binding was observed at pH 6.5 for both supports. As presented in Figure 3B, the laccase immobilization capacities of the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads were 75.1 and 33.8 mg/g beads, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Langmuir isotherm model equation was used to analyze experimental data.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The isotherm equation is: q\u003csub\u003ee\u003c/sub\u003e = q\u003csub\u003em\u003c/sub\u003e C\u003csub\u003ee\u003c/sub\u003e / (K\u003csub\u003ed\u003c/sub\u003e +C\u003csub\u003ee\u003c/sub\u003e) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(2)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experimental adsorption isotherm data is shown in Figure 3A. The isotherm model parameters q\u003csub\u003em\u003c/sub\u003e, and K\u003csub\u003ed\u003c/sub\u003e values were calculated and presented in Table 1. The q\u003csub\u003em\u003c/sub\u003e and K\u003csub\u003ed\u003c/sub\u003e values of immobilized laccases were 92.6 and 49.9 mg protein/g and 1.88x10\u003csup\u003e-6\u003c/sup\u003e and 6.56x10\u003csup\u003e-6\u003c/sup\u003e M for the MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA beads, respectively. The correlation coefficients (R\u0026sup2;) of 0.999 and 0.996 represent the homogeneity of the laccase adsorption process on both tested supports, indicating a monolayer binding. The K\u003csub\u003ed\u003c/sub\u003e value indicates the strength of the enzyme\u0026apos;s binding to the support surface. Consequently, the equilibrium binding constant (K\u003csub\u003ea\u003c/sub\u003e=1/K\u003csub\u003ed\u003c/sub\u003e) derived from the semi-reciprocal plot, and the ∆G values were calculated from the associations ∆G\u003csub\u003eo\u003c/sub\u003e = \u0026minus;RT ln K\u003csub\u003ea\u003c/sub\u003e (R is the gas constant (8.314 J mol\u003csup\u003e-1\u003c/sup\u003e K\u003csup\u003e-1\u003c/sup\u003e) and T is the temperature in Kelvin)\u0026nbsp;and found to be \u0026minus;32.7 and \u0026minus;29.6 kJ/mol for the\u0026nbsp;MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA\u0026nbsp;beads. The negative \u0026Delta;G values showed that the immobilization of laccase on the\u0026nbsp;MCB@PDA-HMDA-Cu(II) and MCB@PDA-HMDA\u0026nbsp;beads was a promising process.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eLangmuir isotherm model constants, correlation coefficients, and ∆G values\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"501\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eq\u003csub\u003ee, exp\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(mg/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003eq\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(mg/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003eK\u003csub\u003ed\u003c/sub\u003ex10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e∆G\u003c/p\u003e\n \u003cp\u003e(kJ/mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eMCB@PDA-HMDA-Cu(II)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e87.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e92.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e5.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-32.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eMCB@PDA-HMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e42.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 55px;\"\u003e\n \u003cp\u003e49.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e1.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 56px;\"\u003e\n \u003cp\u003e0.996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e-29.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Covalent immobilization of laccase on the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMCBs@PDA-HMDA-GA\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;beads\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLaccase was also covalently immobilized on the MCB@PDA-HMDA-GA beads after activation with glutaraldehyde. In this immobilization method, a combination reaction can realize between the amino groups of the matrix and the amine or thiol groups of the enzyme molecules. The grafting of support with PDA-HMDA pairs increased surface area and reactive sites for the covalent immobilization reaction. The maximum laccase immobilization yield of laccase on the MCB@PDA-HMDA-GA beads was 51.6 mg/g, respectively. The recovered activity of the immobilized laccase was about 64.8% on the MCB@PDA-HMDA-GA beads. These reported immobilization efficacy and activity recovery are comparable with earlier studies (Bayramoglu et al., 2012, Kamal et al., 2025. For example, fungal laccase and peroxidase were co-immobilized covalently on a 7% (w/v) polyacrylamide hydrogel and used for biodegradation of BPA, the immobilization efficiency was 78.6 % and activity recovery was 33.7% (Kamal et al., 2025). \u0026nbsp;In these immobilization methods, the measured activity of the adsorbed laccase was significantly lower compared to metal chelate interaction and covalently immobilized laccases. It should be noted that, compared to covalent binding and adsorption methods, the metal chelate interaction method for laccase immobilization showed noticeable advantages in augmenting laccase activity and laccase immobilization capacity (Bayramoglu et al., 2012). This could be due to the presence of Cu(II) on the support, which improved retained enzyme activity. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Effect of pH and temperature on the activity of laccase preparations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimal pH values of laccase preparations were investigated in the pH range 3.0\u0026ndash;8.0 (Figure S5A). For the free laccase and MCB@PDA-HMDA-Cu(II)-Lac preparations, the optimal activities were detected at around 6.0. Whereas the optimal activity for covalently immobilized enzyme (i.e., MCB@PDA-HMDA-GA-Lac) was observed at pH 6.5. The catalytic activity of the free laccase compared to all immobilized laccase preparations is more pH sensitive, whereas the shape of the curves of the immobilized laccase preparations was significantly broadened (Figure S5A). The observed optimal activity shifts of the covalently immobilized laccase from the optimum pH to a less acidic region could be due to the multipoint covalent attachment of the laccase on the MCB@PDA-HMDA-GA beads. This could increase the conformational rigidity and make it more stable at pH 6.5, thus exhibiting changed catalytic performance (Rybarczyk et al., 2025; Sadeghzadeh et al., 2020; Bayramoglu et al., 2019). Furthermore, the immobilized laccase preparations showed comparatively high activity over a wider pH range compared to the free enzyme.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe temperatures activity profiles of the free and immobilized laccase preparations are presented in Figure S5B, the activities of the laccase preparations increased with increasing temperature up to 35 \u0026deg;C, then decreased. However, the immobilized laccase preparations exhibited better temperature stability than their free counterparts (Rodriguez et al., 2025; Bayramoglu et al., 2019). These could result from the formation of relatively stable structures established upon immobilization between enzyme molecules and the functional groups of the support. In particular, the immobilization of the enzyme via multipoint interaction may reduce the conformational flexibility, thus improving its activity space and structure, and may render it more stable at the given temperature (Bayramoglu et al., 2018). Similar observation have been also reported previously, for example, Hydroxyapatite-coated magnetic nanoparticles was used as a support for laccase enzyme immobilization and showed increased stability and catalytic activity in a broader pH range and higher temperatures than its free counterpart, with optimal activity at pH 6.0 (El-Shishtawy et al., 2025).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Kinetic parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKinetic parameters of the enzyme preparations, i.e., K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e values, were calculated using syringaldazine as an artificial substrate. The K\u003csub\u003em\u003c/sub\u003e values of all the immobilized laccase preparations (i.e., immobilization via adsorption, metal chelate interactions, and covalent binding) were higher than those of the free enzyme (Table 2). \u0026nbsp;The K\u003csub\u003em\u003c/sub\u003e values of the immobilized laccase preparations (adsorption, metal chelate interactions, and covalent binding) were increased by about 1.77, 1.33, and 2.55-fold compared to the free laccase. The K\u003csub\u003em\u003c/sub\u003e value shows the affinity of the enzyme to its substrate, and a high K\u003csub\u003em\u003c/sub\u003e value indicates a low affinity of the enzyme to the substrate. The increase in the K\u003csub\u003em\u003c/sub\u003e values of the immobilized laccase preparations could be due to deformation of the active site of the immobilized enzyme preparations, resulting from the reduced mobility of the enzyme\u0026apos;s functional groups, which consequently diminishes its affinity to the substrate (Wehaidy et al., 2024).\u003c/p\u003e\n\u003cp\u003eWhereas, the V\u003csub\u003emax\u003c/sub\u003e value of the immobilized laccase preparations was reduced by about 1.25, 1.07, and 1.45-fold compared to the free enzyme. The declined V\u003csub\u003emax\u003c/sub\u003e values of all the immobilized laccase preparations could be due to lower accessibility of the immobilized enzymes to interact with the substrate compared to their free counterparts (Bayramoglu et al., 2019). The V\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e ratio describes the degree of catalytic activity of the enzyme-substrate sets. The catalytic activity (V\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e) of the free and immobilized laccase preparations on the MCB@PDA-HMDA-Lac, MCB@PDA-HMDA-Cu(II)-Lac, and MCB@PDA-HMDA-GA-Lac were calculated as 38.8, 60.6, and 23.3, and the catalytic efficiencies of the immobilized preparation were decreased compared to free laccase by about 2.23, 1.43, and 3.72 folds, respectively. The stability of the covalently immobilized laccase is improved due to the multipoint linkage formation, while decreased the flexibility of enzyme for substrate binding. The efficiency factor \u0026ldquo;\u0026eta;\u0026rdquo; can be determined from the maximum reaction rates of the immobilized enzymes over that of the free enzyme, and the calculated productivity factors for immobilized laccase preparations are presented in Table 2.\u003c/p\u003e\n\u003cp\u003eɳ = ѵ\u003csub\u003ei\u003c/sub\u003e /ѵ\u003csub\u003ef\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(3)\u003c/p\u003e\n\u003cp\u003ewhere v\u003csub\u003ei\u003c/sub\u003e and v\u003csub\u003ef\u003c/sub\u003e are the reaction rates of the immobilized enzyme and free enzyme, respectively. The decrease in the order of the catalytic efficiency factor of immobilized preparations compared to free laccase was MCB@PDA-HMDA-Cu(II)-Lac \u0026gt; MCB@PDA-HMDA-Lac \u0026gt; MCB@PDA-HMDA-GA-Lac. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Kinetic parameters of the free and immobilized laccase\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003eEnzyme preparations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eK\u003csub\u003em\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(mM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(U g\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026eta;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003eFree laccase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e23.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e86.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003eMCB@PDA-HMDA-Lac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e18.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e38.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003eMCB@PDA-HMDA-Cu(II)-Lac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e21.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e60.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003eMCB@PDA-HMDA-GA-Lac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.6.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Reusability, thermal, and storage stabilities of the laccase preparations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reusability performance of the immobilized enzyme is an important factor for large-scale industrial applications (Yu et al., 2025). The covalently immobilized laccase (i.e., MCB@PDA-HMDA-GA-Lac) retained approximately 89.8% of its initial activity after five successive uses (Figure 4A). On the other hand, the\u0026nbsp;MCB@PDA-HMDA-Lac and MCB@PDA-HMDA-Cu(II)-Lac retained about 57.2% and 69.6% of their initial activities, respectively. The excellent reusability of the covalently immobilized laccase may be attributed to the firm fixation of the enzyme to the support, which generates a stable multipoint binding and consequently diminishes the enzyme\u0026apos;s release from the support.\u003c/p\u003e\n\u003cp\u003eThe long-term storage stability of the laccase preparations was studied by storing them in phosphate buffer solution (20 mmol/L, pH 6.5) at 4 \u0026deg;C for 8 weeks. After this period, the free laccase, MCB@PDA-HMDA-Lac, MCB@PDA-HMDA-Cu(II)-Lac, and MCB@PDA-HMDA-GA-Lac lost 89.2%, 67.5%, 54.8% and 32.4% of their initial activities, respectively Figure 4B. The free laccase retained only 13% its initial activity after 56 days of storage at 4 \u0026deg;C. The stability order of the laccase preparation was MCB@PDA-HMDA-GA-Lac \u0026gt; MCB@PDA-HMDA-Cu(II)-Lac \u0026gt; MCB@PDA-HMDA-Lac \u0026gt; free laccase. The covalent immobilization of the enzyme on the MCB@PDA-HMDA-GA-Lac provided a higher shelf life compared to the other laccase preparations. The improved storage stability of the immobilized laccase preparations can be attributed to the microenvironment of the support materials and the enzyme\u0026apos;s fixation via multipoint attachment on these supports, which protects the enzyme from denaturing factors.\u003c/p\u003e\n\u003cp\u003eThe thermal stabilities of the laccase preparations were studied in the absence of substrate at two different temperatures (i.e., 55 \u0026deg;C and 70\u0026deg;C). As observed from Figure S6, the immobilized laccase preparations were inactivated at a much slower rate compared to the free enzyme for the studied temperatures. At 55 \u0026deg;C for 120 min incubation time, the MCB@PDA-HMDA-GA-Lac, and MCB@PDA-HMDA-Cu(II)-Lac, maintained their activities approximately 79.3%, and 58.6%, and whereas the free enzyme preserved its activity about 26.4%. At 70 \u0026deg;C for 120 min, the free enzyme lost all its activity after a 60-minute incubation period, while the MCB@PDA-HDMA-GA-Lac and MCB@PDA-HDMA-Cu(II)-Lac preserved about 44.6, and 21.3, of their initial activities, respectively. These results showed that the stabilities of the immobilized laccase preparations to heat were significantly improved upon immobilization. Similar observation have been also reported in the previous studies (Bayramoglu et al., 2019, Borham et al. (2025). Borham et al. (2025) studied the immobilization of fungal laccase onto red seaweed biomass via glutaraldehyde crosslinking, it exhibited high thermal stability, retaining 31% relative activity at 80◦C, while free enzyme totally lost its activity.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. Enzymatic degradation studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7.1. Effect of the presence of mediator on the enzymatic degradation of BPA and RG-5 dye\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe degradation studies of BPA and RG-5 dye with the free laccase, MCB@PDA-/HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac beads were monitored by taking samples at different time intervals and examined as defined above. As a control, the heat-inactivated immobilized enzyme preparations did not exhibit any degradation activity for BPA and RG-5 dye. However, using active immobilized laccase preparations, substantial changes were detected in the UV\u0026ndash;vis absorbance spectra of BPA and RG-5 dye. The degradation rates of BPA and RG-5 dye in the absence and presence of acetosyringone as a mediator chemical with time were studied using MCB@PDA-HMDA-GA-Lac and MCB@PDA-HMDA-Cu(II)-Lac. The initial concentrations of BPA and RG-5 were 20.0 mg/mL (Figure 5A). As observed from this figure, the degradation rates of both immobilized laccase preparations for BPA and RG-5 dye were significantly enhanced in the presence of acetosyringone compared to their absence. The biodegradation rates of BPA and RG-5 were found to be 98.4% and 78.2% using MCB@PDA-HMDA-Cu(II)-Lac and 83.7% and 67.2% using MCB@PDA-HMDA-GA-Lac, respectively. On the other hand, in the absence of the mediator compound, the degradation performances of MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac were 67.6% and 52.7% for BPA, and 38.3% and 22.8% for RG-5 dye, respectively. As observed from Figure 5A, the degradation performance of the MCB@PDA-HMDA-Cu(II)-Lac preparation for BPA and RG-5 dye was higher for both pollutants compared to MCB@PDA-HMDA-GA-Lac. It should be noted that the free laccase degraded BPA and RG-5 in the presence of the mediator compound, with approximately 37.4% and 13.1% degradation, respectively. In the absence of the mediator, degradation was observed at 21.6% and 9.8%, respectively. These results showed that the immobilized laccase preparations exhibited high performance in degrading both pollutants compared to the free enzyme. These results could be ascribed to enhanced stabilities and increased catalytic performances deliberated by immobilization. It should be noted that the degradation rates of BPA and RG-5 dye in the presence of mediator compound by the MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac were clearly higher than those in the absence of mediator. For all cases, the biodegradation rate of BPA was higher than that of the RG-5 dye for both immobilized laccase preparations, as determined by experiments with a 120-min contact period. Finally, both immobilized laccase preparations exhibited better performance for degradation of BPA and RG-5 dye compared to the free enzyme, and similar observations were stated for the immobilized laccase in the earlier works and many other oxidative enzymes (Bayramoglu et al 2019; Zhank et al., 2022; Zhang et al., 2023; Suhaimi et al., 2025). For examples, Abkenar and Dehnavi (2025) studied the entrapped of laccase enzyme within MIL\u0026ndash;100(Fe) metal-organic frameworks. It was used for removal of BPA, and the removal performance of BPA was reported as 83 % from \u0026nbsp; 20 mg/L concentration. Antanaskovic et al studied the immobilization of laccase on biochar via adsorption, and evaluates its application for brilliant green degradation. More than 92% of brilliant green (50 mg/L) was removed within 4 h, at pH 5 and 30\u0026deg;C.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7.2. Effect of initial concentration of BPA and RG-5 dye on biodegradation rates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial concentrations of BPA and RG-5 solutions were varied in the medium between 5.0 and 30.0 mg/L in the presence of the mediator compound. As presented in Figure 5B, the amount of biodegraded BPA and RG-5 by the free enzyme, MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac progressively increased with increasing initial concentrations of the tested pollutants in the medium. When the initial concentrations of BPA and RG-5 dye were higher than 20 mg/L, the degradation rates did not increase meaningfully. At 20 mg/g BPA concentration, the removal rates of BPA with the free laccase, MCB@PDA-HMDA-Cu(II)-Lac and MCB@PDA-HMDA-GA-Lac were found to be 37.1%, 96.9%, and 77.8%, respectively, while at 20 mg/g RG-5 concentration, the removal rates were observed as 13.2%, 78.2% and 47.6 %, respectively. As observed in Figure 5B, when the initial concentrations of BPA and RG-5 dye were increased, the likelihood of BPA and/or RG-5 dye molecules interacting with the enzyme in the medium increased, thereby enhancing the removal rates. While the initial concentrations of BPA or RG-5 were further increased, it could become toxic to the enzyme molecules. Thus, this could negatively influence the catalytic activity and degradation performance of the enzyme. Finally, the reduction in the degradation performances of all the laccase preparations at high pollutant concentrations could be due to the poisoning effect of BPA and RG-5 dye and their byproducts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8. Determination of BPA and RG-5 dye degradation rates\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewith MALDI-ToF MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe spectra of the BPA and RG-5 dye achieved at the zero-point, and positive ion MALDI mass spectrum of BPA and RG-5 dye at different time intervals are presented in Figure 6 (partly A and B, respectively). In Figure 6A, the mass spectrum of BPA in the medium showed a monoisotopic peak at 251.1 m/z. After treatment with MCB@PDA-HMDA-Cu(II)-Lac beads, a noticeable decrease in the intensity of the BPA peaks was observed as the contact period increased with the MCB@PDA-HMDA-Cu(II)-Lac preparation. From the signal-to-noise ratios, the percentage removal of BPA was estimated, focusing on the signal-to-noise ratio of the protonated molecular ion peak of the BPA molecule. The percentage of enzymatic degradation of BPA was found to be 47.3%, 76.6%, and 97.7% for 30, 60, and 120 minutes, respectively. At this point, it was observed that the percentage degradation was nearly 97.7% when the contact time was 120 minutes. This elimination proposes that the MCB@PDA-HMDA-Cu(II)-Lac was efficiently degraded by BPA. These results provide valuable insight into the proper degradation of BPA through MCB@PDA-HMDA-Cu(II)-Lac oxidation, suggesting a potential approach for environmental remediation applications.\u003c/p\u003e\n\u003cp\u003eAfter contact with MCB@PDA-HMDA-Cu(II)-Lac and RG-5 dye for 120 min, the observed new peaks could be attributed to enzymatic degradation products, such as the diazo side chain and \u0026ndash;NH-bonding (Figure 6B). From signal/noise ratios, the percentage removal of RG-5 was calculated, focusing on the signal/noise ratio of the protonated molecular ion peak of the dye. The percentage removal of RG-5 (10 mg/L) was 27.3%, 53.6%, and 80.2% at 30, 60, and 120 min, respectively. Here, it was observed that the degradation kinetics of RG-5 dye were significantly slower than those of BPA used in this study. This is the case of the stable phthalocyanine core of RG-5 dye. As reported earlier, laccase is a useful biocatalyst for removing many complex aromatic compounds, such as dyes, pesticides, and chlorinated phenols, from aqueous media (Zhang et al., 2025; Zheng et al., 2023; Vallejo et al., 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e3.9. Toxicity studies\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eD. magna\u003c/em\u003e is a small planktonic crustacean extensively spread in pools and lakes in many countries, and utilized in many toxicity test studies. \u003cem\u003eD. magna\u003c/em\u003e is very susceptible to many toxic chemicals and gives a quick response. The effects of BPA and RG-5 dye concentrations on \u003cem\u003eD. magna\u003c/em\u003e mobility were examined for 48\u0026thinsp;h. Up to 5.0\u0026thinsp;mg/L concentrations of pure BPA and/or RG-5 dye did not show any toxic effect on \u003cem\u003eD. magna\u003c/em\u003e. The EC\u003csub\u003e50\u003c/sub\u003e values of pure BPA and RG-5 dye in 48\u0026thinsp;h were found to be 13.8 and 7.2 mg/L, respectively. The obtained results are comparable to those of earlier studies, and the reported LC50 values for 48 h ranged from 3.9 to 20.0 mg/L for D. magna (Bayramoglu et al., 2019; Bae and Freeman, 2007). The higher toxicity of RG-5 dye compared to BPA may be attributed to its complex chemical structure. Generally, a longer contact time results in a smaller EC50 value, allowing for the detection of the chemical\u0026apos;s toxicity more effectively. At an initial concentration of 25 mg/L, BPA and RG-5 dye were treated with the MCB@PDA-HMDA-Cu(II)-Lac preparation for 120 min, and the toxicity of the samples was evaluated using \u003cem\u003eD. magna\u003c/em\u003e. \u0026nbsp;After treatment of BPA and RG-5 for 120 min, the enzymatically treated solution of BPA did not show any toxic effect on \u003cem\u003eD. magna\u003c/em\u003e, while the byproducts of RG-5 dye displayed a toxic effect on \u003cem\u003eD. magna\u003c/em\u003e. The immobility of \u003cem\u003eD. magna\u003c/em\u003e was about 48%.\u003c/p\u003e\n\u003cp\u003eFreshwater algae species are frequently used as bio-indicators for toxicity testing of many chemicals. Among them, \u003cem\u003eChlorella vulgaris\u003c/em\u003e is highly sensitive to numerous chemicals and is commonly used in toxicity studies. The growth inhibition test of BPA and RG-5 dye was performed on C. vulgaris at three different concentrations of BPA and/or RG-5 dye (i.e., 5.0, 10, and 25.0\u0026thinsp;mg/L). The degradation byproducts of the BPA and RG-5 dye were obtained after treatment with the MCB@PDA-HMDA-Cu(II)-Lac preparation for 120 min. The initial concentration of each pollutant was 25 mg/g. These byproducts were also used in the algal growth inhibition test as described above. As shown in Table S1, the reduction in the growth rate of C. vulgaris for BPA and RG-5 dye was found to be dependent on the concentration of these test compounds. An increase in the initial BPA or RG-5 concentration resulted in a reduction in the harvested biomass weight. The maximum harvested biomass of \u003cem\u003eC. vulgaris\u003c/em\u003e in the control culture was 0.817\u0026thinsp;g/L dry weight, achieved after seven days of incubation. At a 25 mg/g BPA concentration, the harvested biomass of \u003cem\u003eC. vulgaris\u003c/em\u003e was considerably reduced and found to be 0.358\u0026thinsp;g/L dry biomass after a seven-day incubation period. The \u003cem\u003eC. vulgaris\u003c/em\u003e grew at low concentrations of BPA and RG-5 dye at 5.0 mg/L. On the other hand, at a concentration of 25 mg/g of RG-5, the amount of biomass harvested decreased significantly to 0.173 mg/g (Table S1), compared to the control (0.817 g/L). According to these observations, experiments conducted with \u003cem\u003eC. vulgaris\u003c/em\u003e and RG-5 dye showed more toxic effects than those of BPA. It should be noted that numerous reports have been published on the toxicity assessments of dyes using various freshwater alga species; however, no report is currently available on the toxicity assessments of RG-5 dye using alga species. After 120 min of enzymatic treatment of BPA and RG-5, there was no inhibition of the growth of algae, while the treated RG-5 sample showed 77% growth inhibition. Therefore, the presented work may provide important information to define the potential risk of RG-5 dye toxicity on the freshwater algal species.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, magnetic cellulose beads MCB were prepared via the extrusion-dropping method and then grafted with PDA-HMDA. The laccase was immobilized via copper-ion coordination or covalently on the MCB@PDA-HMDA beads after Cu(II) chelating or GA activation, respectively. In comparison with MCB@PDA-HMDA-Cu(II)-Lac preparation, the covalently immobilized laccase (i.e., MCB@PDA-HMDA-GA-Lac) displayed high stability and good reusability performance after five runs. On the other hand, the immobilized laccase via metal chelate interaction exhibited high recovered activity and effectively degraded BPA and RG-5 dye compared to the covalently immobilized counterpart. The degradation of BPA and RG-5 was achieved using both free and immobilized laccase preparations in a batch system. The degradation percentage of BPA (20 mg/L) and RG-5 dye (10 mg/L) was obtained after contact with MCB@PDA-HMDA-Cu(II)-Lac in the presence of mediator compounds for 120 min, and up to 98.4% degradation of BPA was observed, whereas RG-5 was degraded about 78.2% after 120 min incubation. While MCBs@PDA-HMDA-GA-Lac degraded about 83.7 and 67.2 % of BPA and RG-5, respectively. The MCB@PDA-HMDA-Cu(II)-Lac preparation showed good degradation performance for both tested pollutants. Biodegradation rates of BPA and RG-5 were also determined using MALDI-TOF-MS spectra. This study also investigated the toxicological properties of BPA and RG-5 dye, providing an understanding of the toxicity risk of BPA and RG-5 on the mobility of \u003cem\u003eD. magna\u003c/em\u003e and \u003cem\u003eC. vulgaris\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGulay Bayramoglu: Methodology, Investigation, Validation, Formal analysis, Resources, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. M. Yakup Arica: Conceptualization, Writing - review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbkenar SMKN, Dehnavi SM (2025) Laccase immobilization onto MIL\u0026ndash;100 (Fe) and chitosan nanofibers: Fabrication, characterization, and investigation of the potential for bisphenol A removal. J Water Process Eng 74: 107827. https://doi.org/10.1016/j.jwpe.2025.107827 \u003c/li\u003e\n\u003cli\u003eAghera DJ, Bhatt K, Thakkar JB, Prabha CR (2025) Fabrication of an efficient hydroquinone biosensor with laccase immobilized on multi-walled carbon nanotubes. Indian J Biochem Biophys 62:46-53. https://doi.org/10.56042/ijbb.v62i1.15015\u003c/li\u003e\n\u003cli\u003eAlokpa K, El‑Yagoubi Y, Cabana H (2025) Development and optimization of a silica‑bound laccase biocatalyst and its application in hospital wastewater treatment. 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J Hazard Mater 443:130372. https://doi.org/10.1016/j.jhazmat.2022.130372\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Magnetic particles, Cellulose beads, Dopamine-polyethyleneimine coating, Laccase, Phenolic compounds, Enzymatic degradation","lastPublishedDoi":"10.21203/rs.3.rs-8013262/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8013262/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCellulose-based materials can be an ideal immobilizing support for enzymes due to their abundance in nature, non-toxicity, sustainability, and biodegradability. Furthermore, they do not leave any toxic residue behind after the process is utilized. In this work, magnetic cellulose beads (MCB) were coated with polydopamine-polyethyleneimine (PDA-HMDA). The MCB@PDA-HDMA beads were then functionalized by chelating Cu(II) ions or reacting with glutaraldehyde (GA) for the immobilization of laccase via metal chelate interaction or covalent binding, respectively. PDA-HMDA polymer pairs were coated onto MCB to enhance Cu(II) ions chelation and GA functionalization, which provided metal chelate interaction and covalent binding sites for laccase. The MCB@PDA-HDMA beads-based preparations were characterized using FT-IR, X-ray diffraction, SEM, TEM, and VSM. The amounts of immobilized laccase via metal chelate interaction, covalent binding, and adsorption on the MCB@PDA-HDMA-Cu(II)-Lac, MCB@PDA-HDMA-GA-Lac, and MCB@PDA-HDMA beads were found to be 87.9, 51.6, and 42.4 mg/g beads, respectively. The highest activity yield order of immobilized laccase preparations was MCB@PDA-HDMA-Cu(II)-Lac (81.6%)\u0026thinsp;\u0026gt;\u0026thinsp;MCB@PDA-HDMA-GA-Lac (68.4%)\u0026thinsp;\u0026gt;\u0026thinsp;MCB@PDA-HDMA (48.7%) compared to the free laccase using syringaldazine as an artificial substrate. The free enzyme, MCB@PDA-HDMA-Cu(II)-Lac, MCB@PDA-HDMA-Lac preparations exhibited their maximum activities at pH 6.0, whereas MCB@PDA-HDMA-GA-Lac showed its maximum activity at 6.5. The maximum activity for all the laccase preparations was obtained at 35\u0026deg;C. Furthermore, the immobilized forms of laccase displayed good performance over a broader pH range and at higher temperatures. The free laccase was wholly inactivated at 70\u0026deg;C after 60 min incubation in the substrate-free medium, while the MCB@PDA-HDMA-GA-Lac\u0026thinsp;\u0026gt;\u0026thinsp;MCB@PDA-HDMA-Cu(II)-Lac\u0026thinsp;\u0026gt;\u0026thinsp;MCB@PDA-HDMA preserved about 44.6, 21.3, and 19.4% of their initial activities, respectively. The biodegradation of two model pollutants, Bisphenol A (BPA) and Reactive Green 5 (RG-5, metal complex dye), with the free enzyme and MCB@PDA-HDMA-Cu(II)-Lac preparation was studied batch vise. In the presence of acetosyringone as a mediator compound in the reaction medium, biodegradation amounts of BPA (at 20 mg/L) and RG-5 dye (10 mg/mL) were detected as 96.9% and 78.2%, respectively, using MCB@PDA-HDMA-Cu(II)-Lac preparation for a 120 min reaction time.\u003c/p\u003e \u003cp\u003eThe MCB@PDA-HDMA-Cu(II)-Lac preparation displayed high biodegradation performance for both tested pollutants compared to the free laccase. Moreover, the MCB@PDA-HDMA-Cu(II)-Lac and MCB@PDA-HDMA-GA-Lac preparations were used for the degradation of BPA and RG-5 in a batch system over five consecutive cycles. These obtained results make the immobilized laccase preparations favorable candidates for many environmental applications, such as wastewaters management and remediation, where improved enzyme performance and reusability are key parameters.\u003c/p\u003e","manuscriptTitle":"Immobilization of laccase on magnetic cellulose beads for enhanced biodegradation of bisphenol A and a metal-complex dye","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 13:50:26","doi":"10.21203/rs.3.rs-8013262/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-31T23:55:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-31T23:16:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-07T09:04:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellulose","date":"2025-11-02T21:33:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellulose","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cels","sideBox":"Learn more about [Cellulose](https://www.springer.com/journal/10570)","snPcode":"10570","submissionUrl":"https://submission.nature.com/new-submission/10570/3","title":"Cellulose","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6e3ae847-7fef-49e4-b0a1-eff823cf91ab","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T12:38:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 13:50:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8013262","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8013262","identity":"rs-8013262","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}