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Research article
Laccase Immobilization on Copper-Magnetic Nanoparticles for Efficient Bisphenol Degradation
Department of Chemical Engineering, Konkuk University, Seoul 05029, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2023; 33(1): 127-134
Published January 28, 2023 https://doi.org/10.4014/jmb.2210.10032
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
The industrial applications of enzymes are primarily limited by their low stability, substrate or solvent tolerance, and reusability. Various strategies, such as enzyme engineering and immobilization, have been adopted to improve stability [1, 2]. Despite the immense effort required in protein engineering, the results are undesirable and lead to only minor gains in enzyme stability [3]. Through immobilization, enzymes can enhance catalytic activity, stability, and reusability [4]. Several approaches for industrial enzyme immobilization have been reported, such as encapsulation within polymeric materials or metal–protein hybrids [5], adsorption on solid supports or membranes [6, 7], covalent immobilization on supports, biomolecules, or polymers [2, 8], and cross-linking mediated by linkers, such as glyoxal and glutaraldehyde [9, 10]. The additional treatment of glutaraldehyde on immobilized enzymes is beneficial to stabilize enzymes on solid supports, minimizing leaching and improving the structural stability of encapsulated enzymes [11]. Enzyme properties, such as residual activity, substrate specificity, and catalytic parameters, including turnover number,
Nanoparticles (NPs) are considered practical supports for enzyme immobilization because of their unique assets: (i) commercial-scale availability, (ii) chemical alteration to provide suitable functional groups on the surface for enzyme binding, (iii) tunable to desirable sizes with high surface areas, modified to better biocompatibility, (iv) high rigidity to retain support stability during immobilization, and (v) magnetic nature for easy separation from the reaction mixture using a magnet over non-magnetic supports [2, 8, 12, 19, 20]. Numerous valuable enzymes, such as dehydrogenase, cellulase, laccase, and lipase, are widely used in industrial and environmental applications [3, 15, 21, 22]. Laccases are an oxidase group of enzymes that contain multi-copper (Cu). It is applied for purposes, such as the oxidation of harmful phenolic and non-phenolic compounds and degradation of pollutants or dyes [1, 23-26]. Bisphenols, such as bisphenol A and F, are extensively used to synthesize epoxy resins and polycarbonate plastics and are detectable in soft drinks and canned beverages or foods [26, 27]. These bisphenols exhibit high toxicity, cause oxidative stress, and have substantial endocrine-altering potential, especially reproductive and carcinogenic abilities [28, 29]. Therefore, degradation of bisphenols is required to minimize their harmful effects on aquatic and terrestrial organisms, including humans. Laccases can potentially degrade these bisphenols, but this is limited by the low degradation efficiency or stability of free enzyme forms [26, 29]. Various supports, including chitosan, sepiolite, silica, and magnetic NPs, were previously used for laccase immobilization [28-31]. Compared to other systems, the Cu metals-based encapsulation of laccases via metal-protein hybrids is beneficial to retain better residual activity due to the presence of Cu as an active center in laccase [16, 23, 32]. In addition, Cu-based hybrids also exhibit laccase-mimicking properties and are helpful in the degradation of phenolics [33, 34]. However, the Cu-containing magnetic NPs have never been used to immobilize laccase covalently. Therefore, in this study, laccase was immobilized on Cu and Cu-magnetic (Cu/Fe2O4) NPs functionally activated by 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to achieve better activity and easy separation.
Materials and Methods
Chemicals and Materials
APTES, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Cu, Cu/Fe2O4 NPs, glutaraldehyde, and laccase were obtained from Sigma-Aldrich (USA). All other reagents and chemicals were of analytical grade.
Functionalization of Nanoparticles and Laccase Immobilization
The NPs (100 mg) were activated with APTES (2%, v/v) in toluene by incubation for 12 h at room temperature (RT; 25°C) [12]. Glutaraldehyde (1 M) treatment after APTES was performed to provide aldehyde groups on the surface of the NPs at RT for 2 h in 100 mM phosphate buffer (pH 7) [30]. Initially, laccase was immobilized on various functionally activated NPs at pH 7.0 and incubated at 4°C for 24 h using 100 mg of protein/g for support. For efficient laccase immobilization on Cu/Fe2O4 NPs, the optimization parameters, including pH (4.0–8.0), temperature (4–16°C), the incubation period (6–32 h), and enzyme loading (50–600 mg of protein/g supports) were assessed. After immobilization, the NPs were separated by centrifugation (10,000 ×
Activity Assessment
Laccase activity was assessed spectrophotometrically at 420 nm (
Immobilized Laccase Characterization and Kinetic Studies
The characteristics of Cu/Fe2O4-laccase were evaluated using the immobilized enzyme by field-emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) microscopy measurements [4, 23]. The decomposition of Cu/Fe2O4 and Cu/Fe2O4-laccase was compared to validate the high enzyme loading by thermogravimetric analysis (TGA) measurements [7]. The effect of pH on enzyme activity was evaluated in 100 mM of various buffers: glycine-HCl (2.5), sodium citrate (3.0–4.0), and sodium acetate (4.5–6.0). At optimum pH, the influence of temperature (25–70°C) on activity was compared between free and immobilized forms. The kinetic studies were performed using ABTS (0.05–20.0 mM) under standard assay conditions at 25°C in a 100 mM buffer. Furthermore,
Stability and Reusability Measurements
Initially, the storage stability of the enzyme at 4 and 25°C was evaluated for incubation for up to 30 d by measuring residual activity under standard assay conditions. Next, the thermal stability was assessed by incubating enzymes at various temperatures (40–70°C). The reusability of Cu/Fe2O4-laccase was assessed under standard assay conditions for up to 10 recycling cycles. After the first cycle, the Cu/Fe2O4-laccase was collected by centrifugation (10,000 ×
Bisphenols Degradation
Initially, the free and Cu/Fe2O4-laccase were assessed for their degradation ability of bisphenol A and F after 24 h of incubation using a concentration of 50 μM and 2 U/ml of enzyme at 25°C [36]. The residual bisphenol content was analyzed spectrophotometrically using a 4-AAP coupled reaction [35]. Furthermore, the degradation of bisphenols was evaluated at various concentrations (50–250 μM) after incubation for 12 h.
Results and Discussion
Laccase Immobilization on Cu/Fe2O4 NPs
Free
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Table 1 . Laccase immobilization on copper (Cu) and Cu-magnetic (Cu/Fe2O4) nanoparticles (NPs).
NPs Functional activation Immobilization yield (%) Relative activity (%) Cu/Fe2O4 Glutaraldehyde 82.5 ± 4.6 121 ± 10.2 APTES 51.2 ± 4.2 115 ± 8.9 APTES + glutaraldehyde 93.1 ± 3.3 140 ± 10.8 Cu Glutaraldehyde 78.4 ± 4.8 125 ± 10.6 APTES 45.0 ± 4.1 122 ± 9.5 APTES + glutaraldehyde 90.2 ± 3.9 136 ± 11.3 The enzyme immobilization was performed in phosphate buffer (100 mM, pH 7) with a loading of 100 mg of protein/g of support for incubation of 24 h at 4°C.
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Fig. 1. Influence of pH (A), temperature (B), incubation period (C), and loading of laccase on coppermagnetic nanoparticles. IY, Immobilization yield; RA, relative activity.
The loading of enzymes on a support is an essential parameter for assessing the success of immobilization [2]. The maximum laccase immobilization on Cu/Fe2O4 NPs was 285 mg/g of support at a loading rate of 600 mg of protein/g of support, which may be associated with the smaller size of NPs (Fig. 1D). Remarkably, at maximum loading, laccase exhibited a higher RA of 105% than the free form of the enzyme. Previously, a much lower loading of various laccases was reported on i) 14.2 mg/g of composite graphene oxide/CuFe2O4 composite NPs for
Characterization of Immobilized Laccase
FE-SEM analysis confirmed efficient laccase binding by the thick, rough texture of the NP surfaces after immobilization (Figs. 2 A and 2B). Furthermore, laccase immobilization on Cu/Fe2O4 NPs was validated by FTIR analysis in the wavenumber ranges of 500–4,000 cm-1 (Fig. 2C). The peaks between 550 and 600 cm-1 vibrations were associated with Fe-O. In addition, the peaks at 1385 and 1590 cm-1 peaks corresponded to antisymmetric and symmetric carboxylic group stretching, respectively. The broad peaks at 1050, 1230, and 3425 cm-1 represented the stretching vibrations of the alkoxy, epoxy, and hydroxyl groups, respectively. Comprehensive C=O stretching peaks at 1,650 cm-1 (amide I band) and N−H bending at 1,550 (amide II band) vibrations noted in the range of 1,550–1,650 cm-1 validated the immobilization of laccase on the Cu/Fe2O4 NPs. Effective laccase immobilization on Cu/Fe2O4 was assessed by TGA (Fig. 2D). Pure Cu/Fe2O4 exhibited a nearly 8.9% reduction in weight loss at 600°C. Under similar conditions, a high weight reduction of 34.6% for Cu/Fe2O4-laccase validated a maximum NP loading of 285 mg/g.
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Fig. 2. Characterization of laccase immobilized on copper-magnetic (Cu/Fe2O4) nanoparticles (NPs): Field emission scanning electron microscopy images of pure NPs (A), and immobilized laccase (B), Fourier transform infrared microscopy (C), and thermogravimetric (D) analysis.
Enzyme properties, such as the pH and temperature profile, are significantly altered after immobilization [37]. Initially, free and Cu/Fe2O4-laccase activities were compared at a pH range of 2.5–6.0 (Fig. 3A). The free laccase exhibited an optimum pH of 3.5, with residual activity of 52.8% at pH 2.5 and 3.9% at pH 6.0. Immobilized laccase on Cu/Fe2O4 showed a higher optimum pH of 4.0 and higher retention of residual acidity of 1.4- and 10.7-fold at pH 2.5 and 6.0, respectively, than the free enzyme. The optimum pH for laccase was highly variable, depending on the type of substrates, such as ABTS, 2,6-dimethoxyphenol, and 4-phenylenediamine [12, 30, 37]. Previously, no shift in optimum pH was reported for
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Fig. 3. Laccase immobilized on copper-magnetic nanoparticles activity profiles at various pH (A), and temperature (B).
The kinetic parameters
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Table 2 . Kinetic parameters of free and immobilized enzyme on Cu/Fe2O4 NPs.
Laccase K m (mM)V max (μmol/min/mg protein)Free enzyme 1.72 ± 0.38 68.3 ± 6.2 Immobilized enzyme 1.23 ± 0.31 95.6 ± 8.7 The kinetics parameters were measured using ABTS (0.05–20.0 mM) at 25°C in sodium citrate buffer (100 mM) at optimum pH of free and immobilized laccase.
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Table 3 . Immobilization and kinetic parameters of laccases on various supports.
Supports Laccase source Immobilization yield (%) Relative activity (%) K m (mM)V max (μmol/min/mg protein)Reference Cu-alginate Trametes versicolor -a 88.8 0.56 (2.21)c 44.6 (5.4) [16] Chitosan Rhus vernicifera 56.0 30.0 - - [9] Graphene oxide/ CuFe2O4 T. versicolor 14.2b 88.0 1.80 (1.30) 26.0 (56.0) mM/min [39] Nylon membrane R. vernicifera - 2.80 11.3 (69.0) 0.27 (9.58) [6] Silica T. versicolor 75.8 92.9 0.046 (0.029) 1630 (1890) [30] Titania Pleurotus ostreatus 0.80b 126 0.043 (0.037) 101 (75.5) [19] Fe3O4@MoS2 -a 4.70 80.0 58.4 (58.1) 30.3 (31.8) mM/min [27] Fe3O4 T. versicolor 49.0 45.3 0.065 (0.029) 1140 (1890) [7] Cu/Fe2O4 R. vernicifera 93.8 140 1.23 (1.72) 95.6 (68.3) This study aNot available or applicable; bamount of enzyme immobilized in mg/g of support; cvalues within parenthesis are of free enzyme.
Stability and Reusability
The principal objective of immobilization is to achieve improved enzyme stability [10, 13]. The strength of enzymes is highly altered, primarily depending on the immobilization procedures or support properties, such as surface area, morphology, and porosity. The thermostabilities of free and Cu/Fe2O4-laccase were compared at 60°C (Fig. 4A). Free laccase exhibited a progressive decline in residual activity over an incubation period of up to 60 min, and a 96.7% reduction in the residual activity was observed. Under similar conditions, Cu/Fe2O4-laccase retained a high residual activity of 73.4%, and the thermostability of the immobilized laccase was enhanced by up to 25-fold. Previously, a lower improvement of up to 3-fold in thermostability at 60°C was reported after immobilization of various laccases from
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Fig. 4. Stability measurement of laccase immobilized on copper-magnetic nanoparticles at 4°C (A), thermostability at 60°C (B), and reusability (C).
The immobilization of enzymes on magnetic supports is beneficial for their easy separation in the presence of a magnetic field over nonmagnetic supports [36, 39, 42]. Here, the reusability of Cu/Fe2O4-laccase was assessed under standard assay conditions using ABTS for up to 10 cycles (Fig. 4C). Cu/Fe2O4-laccase showed a minor, gradual decline in residual activity after consecutive operations, which might be associated with the leaching of NPs or partial inactivation of the enzyme during recycling. Moreover, Cu/Fe2O4-laccase retained much higher residual activity (96.3% after five cycles and 91.8% after 10 cycles of reuse), which can be associated with the supportive role of Cu in NPs on laccase activity. After 10 cycles of reusability, various immobilized laccases had lower residual activity: i) 21% for
Degradation of Bisphenols
The degradation of bisphenols F and A by free laccase was 58.9 and 71.4%, respectively, after 24 h of incubation with an initial bisphenol concentration of 50 μM (Figs. 5A and 5B). Under similar conditions, Cu/Fe2O4-laccase showed better degradation of 91.4% for bisphenol F and 99.3% for bisphenol A. A lower maximum bisphenol A degradation of up to 68% was observed for laccase immobilized on poly(amidoisophthalic acid)- and cyclodextrin-anchored-based Fe3O4 NPs [28]. Furthermore, the degradation of these bisphenols was evaluated at higher concentrations of up to 250 μM (Figs. 5C and 5D). The degradation of these bisphenols declined at increased concentrations (250 μM) by free and Cu/Fe2O4-laccase. At a bisphenol concentration of 250 μM, a maximum degradation of 14.2 and 24.9% by free laccase was achieved for bisphenol F and bisphenol A, respectively. In contrast, Cu/Fe2O4-laccase exhibited a much higher maximum degradation of 55.4% for bisphenol F and 71.4% for bisphenol A. After immobilization, laccase on Cu/Fe2O4 NPs showed 2.9- and 3.9-fold improvement in the degradation of bisphenol A and bisphenol F, respectively. Previously, bacterial laccase immobilized on magnetic carbon NPs showed a lower improvement (1.5-fold) in bisphenol A degradation [29]. Similarly, an enhancement in the degradation of only 3% for bisphenol A and 1.5-fold for bisphenol F was reported for laccase immobilized on polyethyleneimine-modified Fe3O4@MoS2 core-shell NPs [27].
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Fig. 5. Bisphenol A degradation by laccase immobilized on copper-magnetic nanoparticles profile (A), various concentrations (B), and reusability (C). The degradation of bisphenols was performed by free and immobilized enzymes at 25°C.
Thus, here laccase was immobilized on Cu/Fe2O4 NPs to improve its residual activity, stability, and potential biotechnological applications. After immobilization on Cu/Fe2O4 NPs functionalized by APTES followed by glutaraldehyde, laccase exhibited a higher IY and better RA than the free enzyme. The immobilized laccase showed much better activity profiles over broad pH and temperature ranges. Moreover, a significant increase in the stability of laccase, with high reusability after 10 cycles of reuse, was achieved after immobilization. Cu/Fe2O4-laccase showed significantly higher degradation of bisphenols than the free enzyme. To the best of our knowledge, this is the first report of laccase (
Acknowledgments
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2019R1C1C11009766, 2021R1I1A1A01060963, and 2021H1D3A2A01099705). This work was also supported by the KU Research Professor Program of Konkuk University.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2023; 33(1): 127-134
Published online January 28, 2023 https://doi.org/10.4014/jmb.2210.10032
Copyright © The Korean Society for Microbiology and Biotechnology.
Laccase Immobilization on Copper-Magnetic Nanoparticles for Efficient Bisphenol Degradation
Sanjay K. S. Patel, Vipin C. Kalia, and Jung-Kul Lee*
Department of Chemical Engineering, Konkuk University, Seoul 05029, Republic of Korea
Correspondence to:Jung-Kul Lee, jkrhee@konkuk.ac.kr
Abstract
Laccase activity is influenced by copper (Cu) as an inducer. In this study, laccase was immobilized on Cu and Cu-magnetic (Cu/Fe2O4) nanoparticles (NPs) to improve enzyme stability and potential applications. The Cu/Fe2O4 NPs functionally activated by 3-aminopropyltriethoxysilane and glutaraldehyde exhibited an immobilization yield and relative activity (RA) of 93.1 and 140%, respectively. Under optimized conditions, Cu/Fe2O4 NPs showed high loading of laccase up to 285 mg/g of support and maximum RA of 140% at a pH 5.0 after 24 h of incubation (4°C). Immobilized laccase, as Cu/Fe2O4-laccase, had a higher optimum pH (4.0) and temperature (45°C) than those of a free enzyme. The pH and temperature profiles were significantly improved through immobilization. Cu/Fe2O4-laccase exhibited 25-fold higher thermal stability at 65°C and retained residual activity of 91.8% after 10 cycles of reuse. The degradation of bisphenols was 3.9-fold higher with Cu/Fe2O4-laccase than that with the free enzyme. To the best of our knowledge, Rhus vernicifera laccase immobilization on Cu or Cu/Fe2O4 NPs has not yet been reported. This investigation revealed that laccase immobilization on Cu/Fe2O4 NPs is desirable for efficient enzyme loading and high relative activity, with remarkable bisphenol A degradation potential.
Keywords: Bisphenol A, copper-magnetic nanoparticle, covalent immobilization, laccase, reusability
Introduction
The industrial applications of enzymes are primarily limited by their low stability, substrate or solvent tolerance, and reusability. Various strategies, such as enzyme engineering and immobilization, have been adopted to improve stability [1, 2]. Despite the immense effort required in protein engineering, the results are undesirable and lead to only minor gains in enzyme stability [3]. Through immobilization, enzymes can enhance catalytic activity, stability, and reusability [4]. Several approaches for industrial enzyme immobilization have been reported, such as encapsulation within polymeric materials or metal–protein hybrids [5], adsorption on solid supports or membranes [6, 7], covalent immobilization on supports, biomolecules, or polymers [2, 8], and cross-linking mediated by linkers, such as glyoxal and glutaraldehyde [9, 10]. The additional treatment of glutaraldehyde on immobilized enzymes is beneficial to stabilize enzymes on solid supports, minimizing leaching and improving the structural stability of encapsulated enzymes [11]. Enzyme properties, such as residual activity, substrate specificity, and catalytic parameters, including turnover number,
Nanoparticles (NPs) are considered practical supports for enzyme immobilization because of their unique assets: (i) commercial-scale availability, (ii) chemical alteration to provide suitable functional groups on the surface for enzyme binding, (iii) tunable to desirable sizes with high surface areas, modified to better biocompatibility, (iv) high rigidity to retain support stability during immobilization, and (v) magnetic nature for easy separation from the reaction mixture using a magnet over non-magnetic supports [2, 8, 12, 19, 20]. Numerous valuable enzymes, such as dehydrogenase, cellulase, laccase, and lipase, are widely used in industrial and environmental applications [3, 15, 21, 22]. Laccases are an oxidase group of enzymes that contain multi-copper (Cu). It is applied for purposes, such as the oxidation of harmful phenolic and non-phenolic compounds and degradation of pollutants or dyes [1, 23-26]. Bisphenols, such as bisphenol A and F, are extensively used to synthesize epoxy resins and polycarbonate plastics and are detectable in soft drinks and canned beverages or foods [26, 27]. These bisphenols exhibit high toxicity, cause oxidative stress, and have substantial endocrine-altering potential, especially reproductive and carcinogenic abilities [28, 29]. Therefore, degradation of bisphenols is required to minimize their harmful effects on aquatic and terrestrial organisms, including humans. Laccases can potentially degrade these bisphenols, but this is limited by the low degradation efficiency or stability of free enzyme forms [26, 29]. Various supports, including chitosan, sepiolite, silica, and magnetic NPs, were previously used for laccase immobilization [28-31]. Compared to other systems, the Cu metals-based encapsulation of laccases via metal-protein hybrids is beneficial to retain better residual activity due to the presence of Cu as an active center in laccase [16, 23, 32]. In addition, Cu-based hybrids also exhibit laccase-mimicking properties and are helpful in the degradation of phenolics [33, 34]. However, the Cu-containing magnetic NPs have never been used to immobilize laccase covalently. Therefore, in this study, laccase was immobilized on Cu and Cu-magnetic (Cu/Fe2O4) NPs functionally activated by 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to achieve better activity and easy separation.
Materials and Methods
Chemicals and Materials
APTES, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Cu, Cu/Fe2O4 NPs, glutaraldehyde, and laccase were obtained from Sigma-Aldrich (USA). All other reagents and chemicals were of analytical grade.
Functionalization of Nanoparticles and Laccase Immobilization
The NPs (100 mg) were activated with APTES (2%, v/v) in toluene by incubation for 12 h at room temperature (RT; 25°C) [12]. Glutaraldehyde (1 M) treatment after APTES was performed to provide aldehyde groups on the surface of the NPs at RT for 2 h in 100 mM phosphate buffer (pH 7) [30]. Initially, laccase was immobilized on various functionally activated NPs at pH 7.0 and incubated at 4°C for 24 h using 100 mg of protein/g for support. For efficient laccase immobilization on Cu/Fe2O4 NPs, the optimization parameters, including pH (4.0–8.0), temperature (4–16°C), the incubation period (6–32 h), and enzyme loading (50–600 mg of protein/g supports) were assessed. After immobilization, the NPs were separated by centrifugation (10,000 ×
Activity Assessment
Laccase activity was assessed spectrophotometrically at 420 nm (
Immobilized Laccase Characterization and Kinetic Studies
The characteristics of Cu/Fe2O4-laccase were evaluated using the immobilized enzyme by field-emission scanning electron microscopy (FE-SEM) and Fourier transform infrared (FTIR) microscopy measurements [4, 23]. The decomposition of Cu/Fe2O4 and Cu/Fe2O4-laccase was compared to validate the high enzyme loading by thermogravimetric analysis (TGA) measurements [7]. The effect of pH on enzyme activity was evaluated in 100 mM of various buffers: glycine-HCl (2.5), sodium citrate (3.0–4.0), and sodium acetate (4.5–6.0). At optimum pH, the influence of temperature (25–70°C) on activity was compared between free and immobilized forms. The kinetic studies were performed using ABTS (0.05–20.0 mM) under standard assay conditions at 25°C in a 100 mM buffer. Furthermore,
Stability and Reusability Measurements
Initially, the storage stability of the enzyme at 4 and 25°C was evaluated for incubation for up to 30 d by measuring residual activity under standard assay conditions. Next, the thermal stability was assessed by incubating enzymes at various temperatures (40–70°C). The reusability of Cu/Fe2O4-laccase was assessed under standard assay conditions for up to 10 recycling cycles. After the first cycle, the Cu/Fe2O4-laccase was collected by centrifugation (10,000 ×
Bisphenols Degradation
Initially, the free and Cu/Fe2O4-laccase were assessed for their degradation ability of bisphenol A and F after 24 h of incubation using a concentration of 50 μM and 2 U/ml of enzyme at 25°C [36]. The residual bisphenol content was analyzed spectrophotometrically using a 4-AAP coupled reaction [35]. Furthermore, the degradation of bisphenols was evaluated at various concentrations (50–250 μM) after incubation for 12 h.
Results and Discussion
Laccase Immobilization on Cu/Fe2O4 NPs
Free
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Table 1 . Laccase immobilization on copper (Cu) and Cu-magnetic (Cu/Fe2O4) nanoparticles (NPs)..
NPs Functional activation Immobilization yield (%) Relative activity (%) Cu/Fe2O4 Glutaraldehyde 82.5 ± 4.6 121 ± 10.2 APTES 51.2 ± 4.2 115 ± 8.9 APTES + glutaraldehyde 93.1 ± 3.3 140 ± 10.8 Cu Glutaraldehyde 78.4 ± 4.8 125 ± 10.6 APTES 45.0 ± 4.1 122 ± 9.5 APTES + glutaraldehyde 90.2 ± 3.9 136 ± 11.3 The enzyme immobilization was performed in phosphate buffer (100 mM, pH 7) with a loading of 100 mg of protein/g of support for incubation of 24 h at 4°C..
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Figure 1. Influence of pH (A), temperature (B), incubation period (C), and loading of laccase on coppermagnetic nanoparticles. IY, Immobilization yield; RA, relative activity.
The loading of enzymes on a support is an essential parameter for assessing the success of immobilization [2]. The maximum laccase immobilization on Cu/Fe2O4 NPs was 285 mg/g of support at a loading rate of 600 mg of protein/g of support, which may be associated with the smaller size of NPs (Fig. 1D). Remarkably, at maximum loading, laccase exhibited a higher RA of 105% than the free form of the enzyme. Previously, a much lower loading of various laccases was reported on i) 14.2 mg/g of composite graphene oxide/CuFe2O4 composite NPs for
Characterization of Immobilized Laccase
FE-SEM analysis confirmed efficient laccase binding by the thick, rough texture of the NP surfaces after immobilization (Figs. 2 A and 2B). Furthermore, laccase immobilization on Cu/Fe2O4 NPs was validated by FTIR analysis in the wavenumber ranges of 500–4,000 cm-1 (Fig. 2C). The peaks between 550 and 600 cm-1 vibrations were associated with Fe-O. In addition, the peaks at 1385 and 1590 cm-1 peaks corresponded to antisymmetric and symmetric carboxylic group stretching, respectively. The broad peaks at 1050, 1230, and 3425 cm-1 represented the stretching vibrations of the alkoxy, epoxy, and hydroxyl groups, respectively. Comprehensive C=O stretching peaks at 1,650 cm-1 (amide I band) and N−H bending at 1,550 (amide II band) vibrations noted in the range of 1,550–1,650 cm-1 validated the immobilization of laccase on the Cu/Fe2O4 NPs. Effective laccase immobilization on Cu/Fe2O4 was assessed by TGA (Fig. 2D). Pure Cu/Fe2O4 exhibited a nearly 8.9% reduction in weight loss at 600°C. Under similar conditions, a high weight reduction of 34.6% for Cu/Fe2O4-laccase validated a maximum NP loading of 285 mg/g.
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Figure 2. Characterization of laccase immobilized on copper-magnetic (Cu/Fe2O4) nanoparticles (NPs): Field emission scanning electron microscopy images of pure NPs (A), and immobilized laccase (B), Fourier transform infrared microscopy (C), and thermogravimetric (D) analysis.
Enzyme properties, such as the pH and temperature profile, are significantly altered after immobilization [37]. Initially, free and Cu/Fe2O4-laccase activities were compared at a pH range of 2.5–6.0 (Fig. 3A). The free laccase exhibited an optimum pH of 3.5, with residual activity of 52.8% at pH 2.5 and 3.9% at pH 6.0. Immobilized laccase on Cu/Fe2O4 showed a higher optimum pH of 4.0 and higher retention of residual acidity of 1.4- and 10.7-fold at pH 2.5 and 6.0, respectively, than the free enzyme. The optimum pH for laccase was highly variable, depending on the type of substrates, such as ABTS, 2,6-dimethoxyphenol, and 4-phenylenediamine [12, 30, 37]. Previously, no shift in optimum pH was reported for
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Figure 3. Laccase immobilized on copper-magnetic nanoparticles activity profiles at various pH (A), and temperature (B).
The kinetic parameters
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Table 2 . Kinetic parameters of free and immobilized enzyme on Cu/Fe2O4 NPs..
Laccase K m (mM)V max (μmol/min/mg protein)Free enzyme 1.72 ± 0.38 68.3 ± 6.2 Immobilized enzyme 1.23 ± 0.31 95.6 ± 8.7 The kinetics parameters were measured using ABTS (0.05–20.0 mM) at 25°C in sodium citrate buffer (100 mM) at optimum pH of free and immobilized laccase..
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Table 3 . Immobilization and kinetic parameters of laccases on various supports..
Supports Laccase source Immobilization yield (%) Relative activity (%) K m (mM)V max (μmol/min/mg protein)Reference Cu-alginate Trametes versicolor -a 88.8 0.56 (2.21)c 44.6 (5.4) [16] Chitosan Rhus vernicifera 56.0 30.0 - - [9] Graphene oxide/ CuFe2O4 T. versicolor 14.2b 88.0 1.80 (1.30) 26.0 (56.0) mM/min [39] Nylon membrane R. vernicifera - 2.80 11.3 (69.0) 0.27 (9.58) [6] Silica T. versicolor 75.8 92.9 0.046 (0.029) 1630 (1890) [30] Titania Pleurotus ostreatus 0.80b 126 0.043 (0.037) 101 (75.5) [19] Fe3O4@MoS2 -a 4.70 80.0 58.4 (58.1) 30.3 (31.8) mM/min [27] Fe3O4 T. versicolor 49.0 45.3 0.065 (0.029) 1140 (1890) [7] Cu/Fe2O4 R. vernicifera 93.8 140 1.23 (1.72) 95.6 (68.3) This study aNot available or applicable; bamount of enzyme immobilized in mg/g of support; cvalues within parenthesis are of free enzyme..
Stability and Reusability
The principal objective of immobilization is to achieve improved enzyme stability [10, 13]. The strength of enzymes is highly altered, primarily depending on the immobilization procedures or support properties, such as surface area, morphology, and porosity. The thermostabilities of free and Cu/Fe2O4-laccase were compared at 60°C (Fig. 4A). Free laccase exhibited a progressive decline in residual activity over an incubation period of up to 60 min, and a 96.7% reduction in the residual activity was observed. Under similar conditions, Cu/Fe2O4-laccase retained a high residual activity of 73.4%, and the thermostability of the immobilized laccase was enhanced by up to 25-fold. Previously, a lower improvement of up to 3-fold in thermostability at 60°C was reported after immobilization of various laccases from
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Figure 4. Stability measurement of laccase immobilized on copper-magnetic nanoparticles at 4°C (A), thermostability at 60°C (B), and reusability (C).
The immobilization of enzymes on magnetic supports is beneficial for their easy separation in the presence of a magnetic field over nonmagnetic supports [36, 39, 42]. Here, the reusability of Cu/Fe2O4-laccase was assessed under standard assay conditions using ABTS for up to 10 cycles (Fig. 4C). Cu/Fe2O4-laccase showed a minor, gradual decline in residual activity after consecutive operations, which might be associated with the leaching of NPs or partial inactivation of the enzyme during recycling. Moreover, Cu/Fe2O4-laccase retained much higher residual activity (96.3% after five cycles and 91.8% after 10 cycles of reuse), which can be associated with the supportive role of Cu in NPs on laccase activity. After 10 cycles of reusability, various immobilized laccases had lower residual activity: i) 21% for
Degradation of Bisphenols
The degradation of bisphenols F and A by free laccase was 58.9 and 71.4%, respectively, after 24 h of incubation with an initial bisphenol concentration of 50 μM (Figs. 5A and 5B). Under similar conditions, Cu/Fe2O4-laccase showed better degradation of 91.4% for bisphenol F and 99.3% for bisphenol A. A lower maximum bisphenol A degradation of up to 68% was observed for laccase immobilized on poly(amidoisophthalic acid)- and cyclodextrin-anchored-based Fe3O4 NPs [28]. Furthermore, the degradation of these bisphenols was evaluated at higher concentrations of up to 250 μM (Figs. 5C and 5D). The degradation of these bisphenols declined at increased concentrations (250 μM) by free and Cu/Fe2O4-laccase. At a bisphenol concentration of 250 μM, a maximum degradation of 14.2 and 24.9% by free laccase was achieved for bisphenol F and bisphenol A, respectively. In contrast, Cu/Fe2O4-laccase exhibited a much higher maximum degradation of 55.4% for bisphenol F and 71.4% for bisphenol A. After immobilization, laccase on Cu/Fe2O4 NPs showed 2.9- and 3.9-fold improvement in the degradation of bisphenol A and bisphenol F, respectively. Previously, bacterial laccase immobilized on magnetic carbon NPs showed a lower improvement (1.5-fold) in bisphenol A degradation [29]. Similarly, an enhancement in the degradation of only 3% for bisphenol A and 1.5-fold for bisphenol F was reported for laccase immobilized on polyethyleneimine-modified Fe3O4@MoS2 core-shell NPs [27].
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Figure 5. Bisphenol A degradation by laccase immobilized on copper-magnetic nanoparticles profile (A), various concentrations (B), and reusability (C). The degradation of bisphenols was performed by free and immobilized enzymes at 25°C.
Thus, here laccase was immobilized on Cu/Fe2O4 NPs to improve its residual activity, stability, and potential biotechnological applications. After immobilization on Cu/Fe2O4 NPs functionalized by APTES followed by glutaraldehyde, laccase exhibited a higher IY and better RA than the free enzyme. The immobilized laccase showed much better activity profiles over broad pH and temperature ranges. Moreover, a significant increase in the stability of laccase, with high reusability after 10 cycles of reuse, was achieved after immobilization. Cu/Fe2O4-laccase showed significantly higher degradation of bisphenols than the free enzyme. To the best of our knowledge, this is the first report of laccase (
Acknowledgments
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2019R1C1C11009766, 2021R1I1A1A01060963, and 2021H1D3A2A01099705). This work was also supported by the KU Research Professor Program of Konkuk University.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

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Table 1 . Laccase immobilization on copper (Cu) and Cu-magnetic (Cu/Fe2O4) nanoparticles (NPs)..
NPs Functional activation Immobilization yield (%) Relative activity (%) Cu/Fe2O4 Glutaraldehyde 82.5 ± 4.6 121 ± 10.2 APTES 51.2 ± 4.2 115 ± 8.9 APTES + glutaraldehyde 93.1 ± 3.3 140 ± 10.8 Cu Glutaraldehyde 78.4 ± 4.8 125 ± 10.6 APTES 45.0 ± 4.1 122 ± 9.5 APTES + glutaraldehyde 90.2 ± 3.9 136 ± 11.3 The enzyme immobilization was performed in phosphate buffer (100 mM, pH 7) with a loading of 100 mg of protein/g of support for incubation of 24 h at 4°C..
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Table 2 . Kinetic parameters of free and immobilized enzyme on Cu/Fe2O4 NPs..
Laccase K m (mM)V max (μmol/min/mg protein)Free enzyme 1.72 ± 0.38 68.3 ± 6.2 Immobilized enzyme 1.23 ± 0.31 95.6 ± 8.7 The kinetics parameters were measured using ABTS (0.05–20.0 mM) at 25°C in sodium citrate buffer (100 mM) at optimum pH of free and immobilized laccase..
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Table 3 . Immobilization and kinetic parameters of laccases on various supports..
Supports Laccase source Immobilization yield (%) Relative activity (%) K m (mM)V max (μmol/min/mg protein)Reference Cu-alginate Trametes versicolor -a 88.8 0.56 (2.21)c 44.6 (5.4) [16] Chitosan Rhus vernicifera 56.0 30.0 - - [9] Graphene oxide/ CuFe2O4 T. versicolor 14.2b 88.0 1.80 (1.30) 26.0 (56.0) mM/min [39] Nylon membrane R. vernicifera - 2.80 11.3 (69.0) 0.27 (9.58) [6] Silica T. versicolor 75.8 92.9 0.046 (0.029) 1630 (1890) [30] Titania Pleurotus ostreatus 0.80b 126 0.043 (0.037) 101 (75.5) [19] Fe3O4@MoS2 -a 4.70 80.0 58.4 (58.1) 30.3 (31.8) mM/min [27] Fe3O4 T. versicolor 49.0 45.3 0.065 (0.029) 1140 (1890) [7] Cu/Fe2O4 R. vernicifera 93.8 140 1.23 (1.72) 95.6 (68.3) This study aNot available or applicable; bamount of enzyme immobilized in mg/g of support; cvalues within parenthesis are of free enzyme..
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