Next Article in Journal
Antioxidant Potential and Antibacterial Activities of Caucasian Endemic Plants Sempervivum transcaucasicum and Paeonia daurica subsp. mlokosewitschii Extracts and Molecular In Silico Mechanism Insights
Previous Article in Journal
A Novel HPLC-MS/MS Method for the Intracellular Quantification of the Active Triphosphate Metabolite of Remdesivir: GS-443902
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoX
Volume 15
Issue 4
10.3390/jox15040108
jox-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Immobilization of Pleurotus eryngii Laccase via a Protein–Inorganic Hybrid for Efficient Degradation of Bisphenol A as a Potent Xenobiotic

by
Sanjay K. S. Patel
1,2,
Rahul K. Gupta
1 and
Jung-Kul Lee
1,*
1
Department of Chemical Engineering, Konkuk University, Seoul 05029, Republic of Korea
2
Department of Biotechnology, Hemvati Nandan Bahuguna Garhwal University (A Central University), Srinagar 246174, India
*
Author to whom correspondence should be addressed.
J. Xenobiot. 2025, 15(4), 108; https://doi.org/10.3390/jox15040108
Submission received: 18 May 2025 / Revised: 11 June 2025 / Accepted: 1 July 2025 / Published: 3 July 2025
Browse Figures
Versions Notes

Abstract

In the present investigation, an eco-friendly biocatalyst was developed using Pleurotus eryngii laccase (PeLac) through a copper (Cu)-based protein–inorganic hybrid system for the degradation of bisphenol A, a representative xenobiotic. After partial purification, the specific activity of crude PeLac was 92.6 U/mg of total protein. Immobilization of PeLac as Cu3(PO4)2–Lac (Cu–PeLac) nanoflowers (NFs) at 4 °C resulted in a relative activity 333% higher than that of the free enzyme. The Cu–PeLac NFs exhibited greater pH and temperature stability and enhanced catalytic activity compared to free laccase. This enhanced activity was validated through improved electrochemical properties. After immobilization, Cu–PeLac NFs retained up to 8.7-fold higher residual activity after storage at 4 °C for 30 days. Free and immobilized laccase degraded bisphenol A by 41.6% and 99.8%, respectively, after 2 h of incubation at 30 °C. After ten cycles, Cu–PeLac NFs retained 91.2% degradation efficiency. In the presence of potent laccase inhibitors, Cu–PeLac NFs exhibited a 47.3-fold improvement in bisphenol A degradation compared to free PeLac. Additionally, the synthesized Cu–PeLac NFs demonstrated lower acute toxicity against Vibrio fischeri than Cu nanoparticles. This study presents the first report of PeLac immobilization through an eco-friendly protein–inorganic hybrid system, with promising potential for degrading bisphenol A in the presence of inhibitors to support sustainable development.

1. Introduction

Laccases, multicopper oxidases belonging to the broader family of blue copper proteins, have garnered significant attention across various industrial sectors due to their ability to catalyze the oxidation of a wide range of substrates—particularly phenols and aromatic amines—by utilizing molecular oxygen as the electron acceptor and producing water as the sole byproduct [1,2]. This enzymatic capability makes laccases ideal candidates for applications such as bioremediation, pulp and paper processing, and the synthesis of novel biomaterials, offering a greener alternative to conventional chemical processes [3,4]. Laccases have been identified in diverse biological sources, including plants, bacteria, fungi, actinomycetes, and insects; however, fungal laccases are the most significant for biotechnological applications [5,6]. Despite their versatility, the practical use of free laccases on a large scale is often constrained by inherent limitations such as susceptibility to denaturation and non-reusability. Enzyme immobilization techniques have emerged as a promising approach to overcome these limitations, providing enhanced stability, reusability, and catalytic efficiency [7,8,9].
For immobilization, selecting an appropriate support matrix plays a critical role. Various immobilization strategies such as physical adsorption, covalent attachment, and encapsulation can be employed to immobilize laccases onto support matrices [10,11,12]. Physical adsorption, a relatively simple and cost-effective method, relies on non-specific interactions between the enzyme and the support surface [13]. Covalent attachment involves the formation of stable chemical bonds between the enzyme and the support, resulting in enhanced enzyme stability and reduced leaching [7]. Encapsulation traps the enzyme within a protective matrix, shielding it from harsh environmental conditions and preventing leakage [14,15]. Despite the advantages of laccase immobilization, several limitations and challenges warrant careful consideration. One of the primary challenges lies in maintaining the enzyme’s activity and substrate accessibility following immobilization [16,17]. The choice of support material can significantly influence the catalytic properties of the immobilized system. The immobilization process may induce conformational changes in the enzyme structure, thereby reducing activity or altering substrate specificity [18,19]. Additionally, mass transfer limitations—caused by restricted diffusion of substrates and products within the support matrix—can impede the overall catalytic efficiency of the immobilized enzyme [14,17]. Furthermore, the cost-effectiveness and scalability of the immobilization process are crucial factors to consider for industrial applications. The support material should be relatively inexpensive and environmentally benign, thereby minimizing the economic impact of the process [7,16]. The immobilization method must also be suitable for large-scale production, ensuring a sufficient supply of immobilized enzyme for practical use. The reusability of the immobilized enzyme is another critical factor that determines the overall economic viability of the process [20,21]. Although nanoparticles (NPs) offer several benefits for enzyme immobilization, their high toxicity remains a major concern for environmental sustainability [7,13]. Recently, integrating inorganic materials with proteins to create hybrid nanoflowers (NFs) has emerged as a promising approach with significant potential in biocatalysis and environmental applications [22,23]. Ge et al. reported the formation of protein–inorganic hybrids using copper (Cu) and various enzymes, including α-lactalbumin, laccase, carbonic anhydrase, and lipase [24]. In this mechanism, NFs are synthesized by incubating Cu or other metals with an enzyme in phosphate buffer via three steps: (i) nucleation and formation of primary crystals, (ii) crystal growth, and (iii) assembly into NFs. The type of metal such as Cu, cobalt (Co), nickel (Ni), zinc (Zn), and barium (Ba), as well as the incubation conditions (temperature and duration), significantly influence laccase properties after immobilization through protein–inorganic hybrids as NFs [8,20,21,23,25].
Bisphenol A, a ubiquitous environmental contaminant, has garnered significant attention due to its potential endocrine-disrupting effects and widespread presence in various consumer products, including polycarbonate plastics, epoxy resins, and thermal paper [26,27]. Its extensive use in the packaging industry, particularly in the lining of canned food products, has raised concerns regarding its potential impact on human health, thereby necessitating comprehensive investigation [28,29]. These concerns stem from the compound’s ability to mimic estrogen, potentially disrupting the endocrine system and leading to adverse health outcomes. It has been demonstrated that exposure to bisphenol A can induce estrogenic effects, which may result in reduced fertility, developmental abnormalities, and an increased risk of cancer. Consequently, there is growing interest in developing effective strategies for the degradation and removal of bisphenol A from the environment [30,31,32]. Laccase has diverse sources, including plants, bacteria, and fungi, with redox potential in the range of ~400 to 800 mV [33]. Therefore, the relatively higher redox potential of fungal laccases over plant and bacterial laccases, with their diverse applications, can be useful for immobilization studies. A few reports are available on the purification with a partial characterization of laccases from Pleurotus eryngii (PeLac) [34,35,36]. Still, protein–inorganic hybrid system-based immobilization of PeLac has not been reported.
In the present study, partially purified laccase from PeLac was used to synthesize Cu-based protein–inorganic hybrids as Cu–PeLac NFs through encapsulation, with the aim of enhancing the stability of the free enzyme and facilitating its application in the degradation of bisphenol A, a potent xenobiotic. Upon encapsulation as NFs, PeLac exhibited significant improvements in pH and temperature activity profiles, kinetic properties, and overall stability, while retaining high reusability. Furthermore, Cu–PeLac NFs were effectively applied for bisphenol A degradation under recycling conditions, even in the presence of potent inhibitors.

2. Materials and Methods

2.1. Materials, Chemicals, and Culture

Cu nanoparticles (Cu-NPs), CuSO4, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), dithiothreitol, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water and phosphate-buffered saline (PBS) were obtained from Life Technologies (Carlsbad, CA, USA). A lyophilized culture of Vibrio fischeri was obtained from Modern Water (New Castle, DE, USA). All other analytical-grade reagents were purchased from commercial suppliers. P. eryngii KCCM 60470 was obtained from the Korean Culture Center of Microorganisms (Seoul, Republic of Korea).

2.2. Production and Partial Purification of PeLac

The culture of P. eryngii was grown on a potato dextrose agar plate. Fully grown mycelia (five disks of approximately 5 mm in diameter) were transferred to 100 mL of potato dextrose broth medium in a 0.5 L conical flask and incubated at 30 °C for five days under shaking conditions (175 rpm) to prepare the pre-culture. Thereafter, 10% (v/v) of the pre-culture and a laccase inducer (0.2 mM CuSO4) were added to 1.0 L of basal medium (pH 5.0), and enzyme activity was monitored over a ten-day incubation period [37]. For partial purification, the culture supernatant was first centrifuged at 6000 rpm (4 °C, 30 min), followed by filtration through a 0.22 µm membrane (Millipore, Bedford, MA, USA), and then ultrafiltration using a 30 kDa cut-off membrane (VivaFlow, Vivascience, Hannover, Germany). Protein concentration was determined using the Bradford method [38]. The partially purified PeLac was stored at 4 °C until further use.

2.3. Immobilization of PeLac as Protein–Inorganic Hybrids

Partially purified PeLac was immobilized using a Cu-based protein–inorganic hybrid system. Briefly, CuSO4 (2.0 mM) and PeLac (0.25 mg/mL) were mixed in 3 mL of PBS (10 mM, pH 7.2) and incubated at 4 °C for 24 h [37]. After incubation, the resulting protein–inorganic hybrid precipitates were separated by centrifugation at 10,000 rpm (4 °C, 5 min), and the residual protein concentration in the supernatant was measured. Encapsulation yield (EY), loading, and relative activity (RA) were calculated as follows (Equations (1)–(3)) [37,39]:
EY = (Amount of immobilized enzyme/Initially added enzyme amount) × 100
Loading = Immobilized laccase/Weight of synthesized hybrids
RA = (Activity of immobilized enzyme/Activity of free enzyme) × 100

2.4. Assessment of Enzyme Activity

PeLac activity was evaluated spectrophotometrically using ABTS (εmax = 3.6 × 104 M−1 cm−1) as a substrate at 420 nm [37].

2.5. Characterization of PeLac-Based Protein–Inorganic Hybrids

The activity profiles of free and immobilized PeLac were assessed at various pH values (2.5–8.0) and temperatures (25–70 °C) using ABTS under standard assay conditions. Kinetic parameters (Km and Vmax) were determined using the Michaelis–Menten model via non-linear regression analysis (Prism 5, GraphPad Software, La Jolla, CA, USA) using ABTS concentrations ranging from 0.01 to 2.0 mM at 25 °C [37]. Further, the activity of Cu–PeLac NFs was evaluated in the presence of various ions (5.0 mM), including NaCl, Ca(OH)2, KCl, CuSO4, and Na2CO3, and solvents such as ethanol and methanol (25%, v/v) under standard assay conditions.

2.6. Stability and Reusability

The storage stability of free and immobilized PeLac was evaluated by incubating samples at 4 °C for 30 days in their optimum pH conditions. Further, the pH stability of PeLac was evaluated at various pH levels (2.5–4.5) for a 5-day incubation period. Thermal stability was assessed by incubating the enzymes at temperatures ranging from 40 °C to 70 °C. The initial residual activity of both free and immobilized PeLac was considered 100%. Additionally, the reusability of Cu–PeLac NFs was evaluated over ten cycles under standard assay conditions [39]. Similarly, the enzyme leaching during recycling was evaluated by protein concentration in the supernatant. The leaching (%) was calculated by the amount of the protein ratios in the supernatant to the immobilized × 100 [39].

2.7. Measurement of Acute Toxicity

The acute toxicity of PeLac-based protein–inorganic hybrids (2.0 mg/mL) and pure Cu-NPs (1.0 mg/mL) was assessed against V. fischeri using an 81.9% basic toxicity test protocol by Microtox analyzer (Model 500, Modern Water, USA). Each sample was prepared with various dilutions, and its osmolarity was adjusted with an osmolarity-adjusting solution given by the manufacturer. The endpoint analysis by the Microtox assay detects the decline in the intensity of light emitted by V. fischeri after 30 min of exposure. The effective concentration (EC) value was represented by EC50, indicating a 50% reduction in V. fischeri luminescence after 30 min of incubation [38].

2.8. Degradation of Bisphenol A

The degradation of bisphenol A (50 μM) was carried out using either free (22 µg/mL) or immobilized PeLac (6.4 µg of protein/mL or 39.3 µg of NFs/mL) in 10 mL of buffer (50 mM) at various pH values (4.0–8.0) at 30 °C for 2 h under shaking conditions (100 rpm) [7]. After incubation, the reaction was terminated by adding 100 µL of HCl (0.5 M) to stop the reaction, and the resulting reaction was immediately centrifuged at 10 rpm for 5 min at 4 °C [40]. The residual bisphenol A in supernatant was analyzed via the 4-AAP coupled reaction in the presence of potassium ferricyanide under alkaline conditions that result in the formation of quinone-type dye using absorbance measurements at 506 nm, spectrophotometrically [7]. The effect of bisphenol A concentration (50–200 μM) on degradation by free and immobilized PeLac was evaluated under optimal pH for 2 h of incubation. Repeated batch degradation was conducted using 50 μM bisphenol A at 30 °C for up to ten cycles. Furthermore, degradation of 50 μM bisphenol A was investigated in the presence of potent inhibitors (0.5 mM). For real application, the degradation potential of bisphenol A (50 µM) by free and Cu–PeLac NFs was evaluated in 50% (v/v) supplementation of tap water and wastewater from the lake of Konkuk University in the buffer solution.

2.9. Instrumental Analysis

Field emission scanning electron microscopy (FE-SEM) images of the hybrids were obtained using an FE-SEM system (JSM-6700F, JEOL, Tokyo, Japan) [37]. Diffraction patterns of PeLac-based protein–inorganic hybrids were analyzed by X-ray diffraction (XRD) using an X’Pert PRO MPD diffractometer (Malvern Panalytical, Malvern, UK) [39]. Confocal laser scanning microscopy (CLSM) analysis of hybrids synthesized using FITC-labeled laccase was conducted using a confocal microscope (Olympus, Center Valley, PA, USA) as described previously [39]. The electrochemical properties of free and PeLac-based hybrids were evaluated in a 10 mL cell by cyclic voltammetry (CV) using an SP-150 potentiostat (BioLogic, Knoxville, TN, USA) with a three-electrode setup comprising a platinum wire (counter electrode), Ag/AgCl (saturated KCl, reference electrode), and a glassy carbon electrode (working electrode) [6]. A volume of 5 µL of free or PeLac (1 mg/mL) was fixed on the GCE electrode by adding nafion solution (5%, v/v) at a ratio of 9:1, and dried out for 1–2 h at room temperature. A prepared working electrode was used to analyze ABTS (0.5 mM) oxidation with a scan rate of 20 mV/s, and the potential between 0.0 and 1.3 V vs. Ag/AgCl [6]. Experimental data are presented as mean values ± standard deviations, and statistical significance was assessed using one-way analysis of variance (α = 0.05) in GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results and Discussion

3.1. PeLac Production and Partial Purification

The production profile of PeLac, with and without CuSO4 (0.2 mM), is shown in Figure S1. The highest laccase activity was observed after seven days of incubation, with 1.9 U/mL in the presence of CuSO4 compared to 0.14 U/mL under control conditions. Previously, P. eryngii var. ferulae was reported to produce approximately 3.0 U/mL of laccase activity [35]. In contrast, P. eryngii KS004 exhibited lower maximum activity, reaching only 0.38 U/mL in the presence of 1.0 mM Cu2+ [36]. After partial purification using 30 kDa ultrafiltration membranes, the specific activity of PeLac increased to 92.6 U/mg of total protein, with a yield of 41.9% and a purification fold of 2.1 (Table S1; Figure S2). A lower specific activity of 47.3 U/mg was previously reported for laccase isoenzymes from P. eryngii partially purified by ultrafiltration [34].

3.2. PeLac Immobilization Using Cu-Based Protein–Inorganic Hybrids

The EY and RA of laccase are strongly influenced by the protein concentration during the synthesis of protein–inorganic hybrids. Accordingly, PeLac immobilization was carried out using CuSO4 (2.0 mM) and enzyme concentrations ranging from 0.01 to 0.50 mg/mL at 4 °C (Table 1). After 24 h of incubation, the synthesized protein–inorganic hybrids denoted as Cu3(PO4)2–PeLac (Cu–PeLac) achieved an EY of 84.7%, a loading of 191 mg/g of hybrids, and an RA of up to 333%. A protein concentration of 0.25 mg/mL was found to be optimal, yielding the highest RA (333%) compared to the free PeLac activity (92.6 U/mg of protein). In contrast, purified Trametes versicolor laccase immobilized as Cu–laccase NFs showed a lower EY of 72.4% [8]. Laccases immobilized via protein–inorganic hybrids exhibited varying RA values depending on the metal used: (i) Zn-based hybrids achieved 86.4% RA [25]; (ii) Cu-based hybrids, ~80.0% RA [41]; (iii) Ni-based hybrids, 75.0% RA [21]; and (iv) Co-based hybrids, 60.9% RA [20]. These findings indicate that Cu–based NFS encapsulation of PeLac is highly effective in achieving high RA after immobilization.
FE-SEM analysis demonstrated that the synthesized Cu–PeLac protein–inorganic hybrids exhibited a flower-like morphology characteristic of NFs, with an average size of approximately 10 µm (Figure 1A). Previously reported protein–inorganic hybrids synthesized using other laccases and different metals showed varying morphologies and sizes: (i) ~50 µm for Cu–laccase hybrids with flower-like morphology [42]; (ii) ~2 µm for Co–laccase hybrids with flower-like aggregates [20]; and (iii) ~25 µm for Zn–laccase hybrids with flake-like morphology [25]. Elemental mapping confirmed the formation of Cu–PeLac hybrids by detecting the presence of Cu, carbon (C), nitrogen (N), and phosphorus (P) (Figure 1B–E). XRD analysis validated the synthesis of the hybrids by showing characteristic peaks consistent with the Cu3(PO4)2·3H2O pattern (JCPDS No. 022-0548) (Figure 1F) [37]. To further confirm PeLac immobilization, hybrids were synthesized using FITC-labeled PeLac and analyzed by CLSM measurements (Figure 1G,H). Green fluorescence emission in CLSM confirmed the presence of PeLac within NFs.

3.3. Characterization of Immobilized PeLac

The pH activity profiles of free and immobilized PeLac are shown in Figure 2. The optimum pH values for free and Cu–PeLac NFs were 4.5 and 5.0, with a specific activity of 92.6 and 308 U/mg of protein, respectively (Figure 2A). After immobilization, PeLac exhibited a broader pH activity range, spanning from pH 2.5 to 8.0. Previous studies have reported that laccases immobilized using Co- and Cu-based NFs displayed pH optima similar to their free enzyme counterparts [20,42]. Cu–PeLac NFs demonstrated a 1.6-fold increase in RA at pH 8.0. The optimum temperatures for free and Cu–PeLac NFs were 50 °C and 55 °C with a specific activity of 163 and 404 U/mg of protein, respectively (Figure 2B). Similarly, Cu- and Co-based protein–inorganic hybrids synthesized using laccases have shown temperature optima comparable to those of the free enzyme [42,43]. Laccase immobilized via metal–organic frameworks also exhibited a similar temperature optimum to that of the free enzyme [27]. Furthermore, increasing the incubation temperature up to 70 °C led to a continuous decline in residual activity for free PeLac, whereas Cu–PeLac NFs retained significantly higher residual activity under identical conditions. At 70 °C, Cu–PeLac NFs showed a 14.2-fold higher residual activity than free PeLac. Overall, immobilized PeLac exhibited broader activity profiles across both pH and temperature conditions. These findings suggest that the properties of laccases from different sources can vary substantially following immobilization as Cu-NFs or other hybrid systems. Moreover, the use of crude rather than purified laccases may offer economic advantages for large-scale applications [20,42,43].
Crude PeLac exhibited a Michaelis–Menten constant (Km) of 118 µM and a maximum velocity (Vmax) of 93.1 µmol/min/mg protein toward ABTS as a substrate (Table 2). Following immobilization, the Km value decreased significantly to 48.8 µM, indicating an increased substrate affinity. Previously, laccase immobilized via Co-based hybrids (laccase@Co3(PO4)2·H NFs) demonstrated a similar affinity to that of free laccase, with a Km of approximately 3.0 µM [21]. In contrast, Zn-based laccase NFs exhibited a ~1.5-fold reduction in affinity relative to free laccase, with a Km of 6.3 µM. The Vmax of PeLac increased by 3.3-fold after immobilization as Cu–PeLac NFs. Comparable Km and Vmax values were previously reported for both free and immobilized laccase using Co-based NFs and ABTS as the substrate [25]. In contrast, Cu–laccase NFs showed a ~2.0-fold reduction in substrate affinity after immobilization, with the Km increasing from 40.3 µM to 82.6 µM [42]. Additionally, Bacillus amyloliquefaciens-derived laccase immobilized as Cu–laccase NFs exhibited 11.1% lower catalytic efficiency than its free counterpart [41]. Electrochemical measurements revealed oxidation current peaks of 0.47 µA and 1.42 µA for free and immobilized PeLac, respectively, during the oxidation of ABTS to ABTS+• at a potential of 0.5 V (Table 2). These results confirm that the enhanced catalytic performance of Cu–PeLac NFs is supported by a ~3.0-fold increase in oxidation current relative to the free enzyme. Additionally, the Cu–PeLac NFs retained residual activity of 91.1–116% in the presence of different common ions (Na+, Ca2+, Cl−, Cu2+, and CO32−) and solvents (ethanol and methanol) (Table S2).

3.4. Toxicity Measurements of NFs

NPs are widely recognized as toxic materials capable of contributing to diverse forms of environmental pollution [44]. The synthesized Cu-NFs exhibited an EC50 value of 876 µg/mL (Table 3). In contrast, pure Cu-NPs demonstrated substantially higher toxicity, with an EC50 of 95.4 µg/mL. This marked difference in acute toxicity may be attributed to the larger size and distinct structural characteristics of Cu-NFs compared to Cu-NPs, which are typically ~50 nm in diameter. These findings suggest that Cu-based NFs are highly biocompatible, requiring a 9.2-fold higher concentration to achieve a 50% reduction in of V. fischeri viability after 30 min of incubation. Previously, nano CuO exhibited an EC50 of 79 µg/mL against V. fischeri, indicating higher acute toxicity [45]. In another study, CuO NPs showed an EC50 value of 258 µg/mL under similar 30 min incubation conditions with V. fischeri [46].

3.5. Stability and Reusability

The thermal stability of free and Cu–PeLac NFs was initially evaluated by incubating each enzyme in its optimal pH buffer at temperatures ranging from 40 °C to 70 °C (Table 4). Under these conditions, the half-life (t1/2) of free PeLac ranged from 0.17 to 13.2 h, whereas Cu–PeLac NFs demonstrated an extended t1/2 of 1.12 to 62.6 h. This represents up to an 11.8-fold improvement in PeLac stability following immobilization. In contrast, Co-based T. versicolor laccase immobilized as aggregate protein–inorganic hybrids exhibited lower residual activity than the free enzyme after incubation at 45 °C [20]. The storage stability of free and immobilized PeLac was also assessed by incubation at 4 °C for 30 days (Figure 3A). During storage, the residual activity of free PeLac progressively declined with increasing storage duration. After 30 days, free PeLac retained only 9.9% of its initial activity. In comparison, Cu–PeLac NFs maintained a significantly higher residual activity of 86.4% under identical conditions. In contrast, Co–laccase protein–inorganic hybrids showed substantially lower stability, only a 1.4-fold improvement after 40 days of incubation compared to the control [20]. Further, on incubation at various pH levels of 2.5–4.5 during storage at 4 °C, the residual activity of Cu–PeLac decreased to 92.3% at pH 2.5 compared to 98.2% at the optimum pH of 4.5 (Figure S3). These findings suggest that the morphological structure of synthesized protein–inorganic hybrids can influence enzyme properties post-immobilization, particularly structure–stability relationships that minimize enzyme leaching and inactivation during recycling [20,42].
Under recycling assay conditions, Cu–PeLac NFs retained 94.6% and 93.3% of their initial activity after five and ten reuse cycles, respectively (Figure 3B). Under similar conditions, the cumulative leaching of 5.4% of immobilized enzyme was observed, which validates the decline in residual activity. In comparison, several immobilized hybrid systems using expensive purified laccases have demonstrated significantly lower reusability under similar conditions, with only ~30% residual activity reported for Cu–laccase NFs after ten cycles [43], ~25–40% for laccases immobilized on Ni/Zn- and Ni/Zn/Co-ferrite particles after five cycles [11], ~40% for Zn–laccase NFs after 12 cycles [42], and ~50% for laccases immobilized using magnetic dendrimer-grafted silica-coated hercynite–copper, Cu–laccase, and Co–laccase NFs after ten cycles [18,20,43].

3.6. Bisphenol A Degradation by Free and Immobilized PeLac

Bisphenol A concentrations in wastewater effluents have been reported to reach up to 370 µg/L (1.6 µM), though these levels can vary significantly among different effluent sources [47]. To evaluate the potential application of laccase in xenobiotic removal, both free and immobilized PeLac were used to degrade bisphenol A. The degradation efficiency of bisphenol A and similar xenobiotics varies considerably depending on the laccase source and environmental conditions, including pH and temperature [10,48,49]. Accordingly, bisphenol A at a concentration of 50 µM was subjected to enzymatic degradation at pH values ranging from 4.5 to 8.0 for 2 h at 30 °C (Figure 4A). Free and immobilized PeLac demonstrated differing efficiencies across the tested pH range. The maximum degradation was 41.6% for free PeLac at pH 4.5 and 99.8% for Cu–PeLac NFs at pH 5.0. Compared to the free form, Cu–PeLac NFs exhibited significantly enhanced degradation capacities—2.8-fold higher at pH 4.0 and 6.6-fold higher at pH 8.0 under identical conditions. These improvements may be attributed to enhanced structural stability and the formation of a favorable microenvironment for catalysis following immobilization [19,30]. Similarly, purified T. versicolor laccase immobilized in Cu–alginate beads achieved 96.1% degradation of 44 µM bisphenol A at pH 5.0 [10]. In contrast, free and immobilized T. versicolor laccase on chitosan-functionalized halloysite nanotubes showed comparable degradation efficiencies of up to 85.0% across a broader pH range (3.0–9.0) after 12 h of incubation [30]. Additionally, laccase immobilized on a cellulose/waste Cu2+–activated carbon composite exhibited less than 80% degradation of bisphenol A over a pH of 3.0–7.0 [19].
When the bisphenol A concentration was increased from 50 to 200 µM, a progressive decline in degradation efficiency was observed for free PeLac, decreasing from 41.6% to 25.4% (Figure 4B). In contrast, Cu–PeLac NFs maintained high degradation efficiency, ranging from 95.1% to 99.8%, across the same concentration range. Under repeated-batch degradation conditions, Cu–PeLac NFs retained 94.0% and 90.2% degradation efficiency after five and ten reuse cycles, respectively (Figure 4C). Previously, purified laccase immobilized onto the defective metal–organic framework PCN-224 showed markedly lower degradation performance—only up to ~10% for bisphenol A concentrations ranging from 2.0 to 88.0 µM [31]. Free laccase exhibited ~2.0-fold higher degradation efficiency than its immobilized counterpart on PCN-224 at similar concentrations. Likewise, T. versicolor laccase immobilized in magnetic gelatin methacryloyl–chitosan hydrogel retained 73.7% degradation efficiency of 44.0 µM bisphenol A after ten reuse cycles [29]. Cu–laccase NFs achieved ~80% degradation of mercaptobenzothiazole after five reuse cycles [42], while B. amyloliquefaciens-derived laccase immobilized as Cu–laccase NFs retained ~60% efficiency for tetracycline degradation after five reuse cycles [41]. Additionally, immobilized laccase on PCN-224 retained ~70% residual degradation of 44.0 µM bisphenol A after five cycles—closely matching the ~70% adsorption efficiency of the PCN-224 material alone [31]. Free enzyme activity is significantly inhibited by substances that limit its utility in various biotechnological applications [50,51]. Therefore, bisphenol A degradation by free and immobilized PeLac was further evaluated in the presence of known laccase inhibitors, including L-cysteine, sodium dodecyl sulfate (SDS), 2-mercaptoethanol, and Fe2+ ions (Figure 4D). The degradation efficiency of free PeLac declined sharply from 41.6% (without inhibitor) to 3.2%, 2.8%, 2.1%, and 1.8% in the presence of SDS, L-cysteine, 2-mercaptoethanol, and Fe2+, respectively. In contrast, Cu–PeLac NFs retained high bisphenol A degradation efficiencies ranging from 81.9% to 86.7% in the presence of these inhibitors. Notably, Cu–PeLac NFs demonstrated up to a 47.3-fold improvement in inhibitor tolerance compared to free PeLac. In the mechanism, the higher stability of Cu–PeLac NFs against these inhibitors might be associated with preservation of structural properties of PeLac after encapsulation or alteration in the microenvironment around the active site [39,50]. Previously, laccase immobilized on a polyacrylonitrile/polyethersulfone support exhibited lower estrogen degradation efficiency (up to 70%) in the presence of various surfactants and metal-based inhibitors [50]. In another study, bisphenol A degradation by Cu–alginate bead-immobilized laccase was reduced by more than 33% when buffer was replaced with real water [10]. Similarly, reduced bisphenol A degradation efficiency was also observed for T. versicolor laccase immobilized in magnetic gelatin methacryloyl–chitosan hydrogel [29]. In the real water samples analysis, a decrease of 8.2% and 12.3% in bisphenol A degradation was noted for free PeLac in tap water and wastewater, respectively, as compared to the control (Figure S4). In contrast, a lower than 1.0% decline in bisphenol A degradation was recorded for Cu–PeLac NFs under similar conditions.

4. Conclusions

The selection of a suitable support material plays a critical role in determining the overall effectiveness of enzyme immobilization, as it significantly influences the structural and catalytic properties of the resulting biocatalyst for diverse biotechnological applications. This study reports, for the first time, the efficient immobilization of PeLac using copper-based protein–inorganic Cu–PeLac NFs for the degradation of bisphenol A in the presence of potent enzyme inhibitors. Following immobilization, the Cu–PeLac NFs exhibited enhanced catalytic performance across a wide pH and temperature range, significantly improved stability, and higher degradation efficiency compared to free PeLac. The Cu–PeLac NFs also retained substantial activity in the presence of known inhibitors, demonstrating enhanced robustness and reusability. Furthermore, the synthesized Cu–PeLac NFs showed superior biocompatibility against V. fischeri relative to Cu-NPs. These findings highlight an eco-friendly strategy for immobilizing crude laccases from diverse biological sources and underscore their potential for broad applications in environmental remediation, particularly in the degradation of xenobiotics.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jox15040108/s1, Figure S1: Pleurotus eryngii laccase production profile with and without CuSO4 (0.2 mM); Figure S2: SDS-PAGE analysis of Pleurotus eryngii crude enzymes: M—Protein marker, lane 1—Enzymes treated with 30 kD spin-column; Figure S3: Storage stability (4 °C) of immobilized PeLac at various pH levels for 5 days; Figure S4: Influence of tap and wastewater supplantation in buffer on degradation of bisphenol by free and Cu–PeLac NFs; Table S1: Partial purification of Pleurotus eryngii laccase; Table S2: The influence of common ions and organic solvents on the activity of Cu-PeLac NFs.

Author Contributions

Conceptualization, S.K.S.P. and J.-K.L.; methodology, S.K.S.P.; software, J.-K.L.; validation, S.K.S.P. and J.-K.L.; formal analysis, R.K.G.; investigation, S.K.S.P.; resources, S.K.S.P. and J.-K.L.; data curation, R.K.G.; writing—original draft preparation, S.K.S.P.; writing—review and editing, S.K.S.P. and J.-K.L.; visualization, S.K.S.P.; supervision, S.K.S.P.; project administration, J.-K.L.; funding acquisition, J.-K.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Science, ICT, and Future Planning (NRF-2022M3A9I3082366, 2022M3A9I5015091, RS-2023-00222078, RS-2024-00351665, RS-2024-00440681).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Acknowledgments

The author would like to thank Vice Chancellor, Hemvati Nandan Bahuguna Garhwal University (A Central University), Srinagar, for providing necessary support.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Suman, S.K.; Malhotra, M.; Kurmi, A.A.; Narani, A.; Bhaskar, T.; Ghosh, S.; Jain, S.L. Jute sticks biomass delignification through laccase-mediator system for enhanced saccharification and sustainable release of fermentable sugar. Chemosphere 2022, 286, 131687. [Google Scholar] [CrossRef] [PubMed]
  2. Himanshu; Behera, B.; Kumari, N.; Maruthi, M.; Singh, R.K.; Saini, J.K. Appraisal of malachite green biodegradation and detoxification potential of laccase from Trametes cubensis. Bioresour. Technol. 2025, 417, 131869. [Google Scholar] [CrossRef]
  3. Aghaee, M.; Salehipour, M.; Rezaei, S.; Mogharabi-Manzari, M. Bioremediation of organic pollutants by laccase-metal–organic framework composites: A review of current knowledge and future perspective. Bioresour. Technol. 2024, 406, 131072. [Google Scholar] [CrossRef] [PubMed]
  4. Thathola, P.; Melchor-Martinez, E.M.; Adhikari, P.; Hernandez-Martinez, S.A.; Pandey, A.; Parra-Saldivar, R. Laccase-mediated degradation of emerging contaminants: Unveiling a sustainable solution. Environ. Sci. Adv. 2024, 3, 1500–1512. [Google Scholar] [CrossRef]
  5. Braunschmid, V.; Binder, K.; Fuerst, S.; Subagia, R.; Danner, C.; Weber, H.; Schwaiger, N.; Nyanhongo, G.S.; Ribitsch, D.; Guebitz, G.M. Comparison of a fungal and a bacterial laccase for lignosulfonate polymerization. Process Biochem. 2021, 109, 207–213. [Google Scholar] [CrossRef]
  6. Patel, S.K.S.; Choi, S.H.; Kang, Y.C.; Lee, J.-K. Large-scale aerosol-assisted synthesis of biofriendly Fe2O3 yolk-shell particles: A promising support for enzyme immobilization. Nanoscale 2015, 8, 6728–6738. [Google Scholar] [CrossRef] [PubMed]
  7. Patel, S.K.S.; Kalia, V.C.; Lee, J.-K. Laccase immobilization on copper-magnetic nanoparticles for efficient bisphenol degradation. J. Microbiol. Biotechnol. 2023, 33, 127–134. [Google Scholar] [CrossRef]
  8. Wang, L.; Yuan, T.; Zhang, Y. Application of laccase-inorganic nanoflowers based time-temperature integrator to real-time monitor the freshness of pasteurized milk. Food Chem. X 2024, 24, 101916. [Google Scholar] [CrossRef]
  9. Vallejo, J.L.; Vallejos, S.; Trigo-Lopez, M.; Garcia, J.M.; Busto, M.D. Optimization and stability of a reusable laccase-polymer hybrid film for the removal of bisphenol A in water. Environ. Technol. Innov. 2025, 38, 104093. [Google Scholar] [CrossRef]
  10. Latif, A.; Maqbool, A.; Sun, K.; Si, Y. Immobilization of Trametes versicolor laccase on Cu-alginate beads for biocatalytic degradation of bisphenol A in water: Optimized immobilization, degradation and toxicity assessment. J. Environ. Chem. Eng. 2022, 10, 107089. [Google Scholar] [CrossRef]
  11. Bîtcan, I.; Petrovici, A.; Pellis, A.; Klebert, S.; Karoly, Z.; Bereczki, L.; Peter, F.; Todea, A. Enzymatic route for selective glycerol oxidation using covalently immobilized laccases. Enzym. Microb. Technol. 2023, 163, 110168. [Google Scholar] [CrossRef]
  12. Sun, H.; Jiang, F.; Yang, X. Enhanced stability of Thermus thermophiles SG0.5JP17-16 laccase immobilized by CuF-MOF and its application in the Congo red decolorization. Biochem. Eng. J. 2024, 209, 109397. [Google Scholar] [CrossRef]
  13. Moayedi, M.; Yousefi, N. Laccase immobilized on reduced graphene oxide sponges for simultaneous adsorption and enzymatic degradation of endocrine disrupting chemicals. Int. J. Biol. Macromol. 2025, 302, 139984. [Google Scholar] [CrossRef]
  14. Lee, H.R.; Chung, M.; Kim, M.I.; Ha, S.H. Preparation of glutaraldehyde-treated lipase inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes. Enzym. Microb. Technol. 2017, 105, 24–29. [Google Scholar] [CrossRef]
  15. Wang, H.; Jin, C.; Li, X.; Ma, J.-X.; Ye, Y.-F.; Tang, L.-X.; Si, J.; Cui, B.-K. A green biocatalyst fabricated by fungal laccase immobilized onto Fe3O4@polyaniline-chitosan nanofibrous composites for the removal of phenolic compounds. Chem. Eng. J. 2025, 507, 160486. [Google Scholar] [CrossRef]
  16. Gutiérrez-Antón, M.; Santiago-Hernández, A.; Rodríguez-Mendoza, J.; Cano-Ramírez, C.; Bustos-Jaimes, I.; Aguilar-Osorio, G.; Campos, J.E.; Hidalgo-Lara, M.E. Improvement of laccase production by Thielavia terrestris Co3Bag1. enhancing the bio-catalytic performance of the native thermophilic TtLacA via immobilization in copper alginate gel beads. J. Fungi 2023, 9, 308. [Google Scholar]
  17. Weng, Y.; Chen, R.; Hui, Y.; Chen, D.; Zhao, C.-X. Boosting enzyme activity in enzyme metal–organic framework composites. Chem Bio Eng. 2024, 1, 99–112. [Google Scholar] [CrossRef] [PubMed]
  18. Rezayaraghi, F.; Jafari-Nodoushan, H.; Mojtabavi, S.; Golshani, S.; Jahandar, H.; Faramarzi, M.A. Hybridization of laccase with dendrimer-grafted silica-coated hercynite-copper phosphate magnetic hybrid nanoflowers and its application in bioremoval of Gemifloxacin. Environ. Sci. Pollut. Res. 2022, 29, 89255–89272. [Google Scholar] [CrossRef]
  19. Li, N.; Yu, F.; Li, H.; Meng, X.; Peng, C.; Sheng, X.; Zhang, J.; Liu, S.; Pimg, Q.; Xiao, H. Cellulose / waste Cu2+-activated carbon composite: A sustainable and green material for boosting laccase activity and degradation of bisphenol A in wastewater. Int. J. Biol. Macromol. 2024, 281, 136121. [Google Scholar] [CrossRef]
  20. Vojdanitalab, K.; Jafari-Nodoushan, H.; Mojtabavi, S.; Shokri, M.; Jahandar, H.; Faramarzi, M. Instantaneous synthesis and full characterization of organic–inorganic laccase-cobalt phosphate hybrid nanoflowers. Sci. Rep. 2022, 12, 9297. [Google Scholar] [CrossRef]
  21. Jafari-Nodoushan, H.; Fazeli, M.R.; Faramarzi, M.A.; Samadi, N. Hierarchically-structured laccase@Ni3(PO4)2 hybrid nanoflowers for antibiotic degradation: Application in real wastewater effluent and toxicity evaluation. Int. J. Biol. Macromol. 2023, 234, 123574. [Google Scholar] [CrossRef]
  22. Li, J.-X.; Chai, T.-Q.; Chen, G.-Y.; Luo, M.-L.; Wan, J.-B.; Yang, F.-Q. A novel dual-ligand copper-based nanoflower for the colorimetric and fluorescence detection of 2,4-dichlorophenol, epinephrine and hydrogen peroxide. Anal. Chim. Acta 2024, 1330, 343298. [Google Scholar] [CrossRef] [PubMed]
  23. Moshiri, F.; Mojtabavi, S.; Momeni, S.A.; Abdolghaffari, A.H.; Jahandar, H.; Faramarzi, M.A. Hierarchically-structured laccase@Ba3(PO4)2 hybrid nanoflowers coating on the urinary foley catheter with high durability, biocompatibility, anti-adhesion activity, and contrasting behavior. Mater. Chem. Phys. 2024, 313, 128777. [Google Scholar] [CrossRef]
  24. Ge, J.; Lei, J.; Zare, R.N. Protein-inorganic hybrid nanoflowers. Nat. Nanotechnol. 2012, 7, 428–432. [Google Scholar] [CrossRef]
  25. Kiani, M.; Mojtabavi, S.; Jafari-Nodoushan, H.; Tabib, S.R.; Hassannejad, N.; Faramarzi, M.A. Fast anisotropic growth of the biomineralized zinc phosphate nanocrystals for a facile and instant construction of laccase@Zn3(PO4)2 hybrid nanoflowers. Int. J. Biol. Macromol. 2022, 204, 520–531. [Google Scholar] [CrossRef]
  26. Latif, A.; Maqbool, A.; Zhou, R.; Arsalan, M.; Sun, K.; Si, Y. Optimized degradation of bisphenol A by immobilized laccase from Trametes versicolor using Box-Behnken design (BBD) and artificial neural network (ANN). J. Environ. Chem. Eng. 2022, 10, 107331. [Google Scholar] [CrossRef]
  27. Sun, H.; Yuan, F.; Jia, S.; Zhang, X.; Xing, W. Laccase encapsulation immobilized in mesoporous ZIF-8 for enhancement bisphenol A degradation. J. Hazard. Mater. 2023, 445, 130460. [Google Scholar] [CrossRef] [PubMed]
  28. Liu, X.; Wang, F.; Xia, C.; You, Q.; Chen, X.; Li, Y.; Lin, W.; Guo, L.; Fu, F. Copper nanoparticles incorporated nitrogen-rich carbon nitride as laccase-like nanozyme for colorimetric detection of bisphenol a released from microplastics. Microchem. J. 2023, 190, 108682. [Google Scholar] [CrossRef]
  29. Du, M.; Liu, J.; Huang, B.; Wang, Q.; Wang, F.; Bi, L.; Ma, C.; Song, M.; Jiang, G. Spatial nanopores promote laccase degradation of bisphenol A and its analogs. Sci. Total Environ. 2023, 901, 166429. [Google Scholar] [CrossRef]
  30. Wang, Z.; Ren, D.; Cheng, Y.; Zhang, X.; Zhang, S.; Chen, W. Immobilization of laccase on chitosan functionalized halloysite nanotubes for degradation of Bisphenol A in aqueous solution: Degradation mechanism and mineralization pathway. Heliyon 2022, 8, e09919. [Google Scholar] [CrossRef]
  31. Li, Z.; Yang, Z.; Shi, Q.; Sun, Y. Removal of bisphenol A by integrated adsorption and biodegradation using immobilized laccase onto defective PCN-224. J. Environ. Chem. Eng. 2023, 11, 110166. [Google Scholar] [CrossRef]
  32. Yang, Y.; Jian, Y.; He, L. High performance persistent organic pollutants removal using stabilized enzyme aggregates over amino functionalized magnetic biochar. J. Hazard. Mater. 2025, 491, 137868. [Google Scholar] [CrossRef]
  33. Singh, G.; Kaur, K.; Puri, S.; Sharma, P. Critical factors affecting laccase-mediated biobleaching of pulp in paper industry. Appl. Microbiol. Biotechnol. 2015, 99, 155–164. [Google Scholar] [CrossRef] [PubMed]
  34. Munoz, C.; Guillen, F.; Martinez, A.T.; Martinez, M.J. Laccase isoenzymes of Pleurotus eryngii: Characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation. Appl. Environ. Microbiol. 1997, 63, 2166–2174. [Google Scholar] [CrossRef] [PubMed]
  35. Guo, C.; Zhao, L.; Wang, F.; Lu, J.; Ding, Z.; Shi, G. β-Carotene from yeasts enhances laccase production of Pleurotus eryngii var. ferulae in co-culture. Front. Microbiol. 2017, 8, 1101. [Google Scholar] [CrossRef]
  36. Sharghi, S.; Ahmadi, F.S.; Kakhki, A.M.; Farsi, M. Copper increases laccase gene transcription and extracellular laccase activity in Pleurotus eryngii KS004. Braz. J. Microbiol. 2024, 55, 111–116. [Google Scholar] [CrossRef]
  37. Patel, S.K.S.; Gupta, R.K.; Kim, I.-W.; Lee, J.-K. Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol. Enzym. Microb. Technol. 2023, 170, 110301. [Google Scholar] [CrossRef]
  38. Patel, S.K.S.; Gupta, R.K.; Karuppanan, K.K.; Padhi, D.K.; Ranganathan, S.; Paramanantham, P.; Lee, J.-K. Trametes versicolor laccase-based magnetic inorganic-protein hybrid nanobiocatalyst for efficient decolorization of dyes in the presence of inhibitors. Materials 2024, 17, 1790. [Google Scholar] [CrossRef]
  39. Patel, S.K.S.; Otari, S.V.; Li, J.; Kim, D.R.; Kim, S.C.; Cho, B.K.; Kalia, V.C.; Kang, Y.C.; Lee, J.-K. Synthesis of cross-linked protein-metal hybrid nanoflowers and its application in repeated batch decolorization of synthetic dyes. J. Hazard. Mater. 2018, 347, 442–450. [Google Scholar] [CrossRef]
  40. Modaressi, K.; Taylor, K.E.; Bewtra, J.K.; Biswas, N. Laccase-catalyzed removal of bisphenol-A from water: Protective effect of PEG on enzyme activity. Water Res. 2005, 39, 4309–4316. [Google Scholar] [CrossRef]
  41. Han, Z.; Wang, H.; Zheng, J.; Wang, S.; Yu, S.; Lu, L. Ultrafast synthesis of laccase copper phosphate hybrid nanoflowers for efficient degradation of tetracycline antibiotics. Environ. Res. 2023, 216, 114690. [Google Scholar] [CrossRef] [PubMed]
  42. Al-Maqdi, K.A.; Elmerhi, N.; Alzamly, A.; Shah, I.; Ashraf, S.S. Laccase–copper phosphate hybrid nanoflower as potent thiazole remediation agent. J. Water Process Eng. 2023, 51, 103438. [Google Scholar] [CrossRef]
  43. Zhang, M.; Zhang, Y.; Yang, C.; Ma, C.; Zhang, Y.; Tang, J. Facile synthesis of recyclable laccase-mineral hybrid complexes with enhanced activity and stability for biodegradation of Evans Blue dye. Int. J. Biol. Macromol. 2021, 188, 783–789. [Google Scholar] [CrossRef] [PubMed]
  44. Chen, F.; Wu, L.; Xiao, X.; Rong, L.; Li, M.; Zou, X. Mixture toxicity of zinc oxide nanoparticle and chemicals with different mode of action upon Vibrio fischeri. Environ. Sci. Eur. 2020, 32, 41. [Google Scholar] [CrossRef]
  45. Heinlaan, M.; Ivask, A.; Blinova, I.; Dubourguier, H.-C.; Kahru, A. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 2008, 71, 1308–1316. [Google Scholar] [CrossRef]
  46. Rossetto, A.L.O.F.; Melegari, S.P.; Ouriques, L.C.; Matias, W.G. Comparative evaluation of acute and chronic toxicities of CuO nanoparticles and bulk using Daphnia magna and Vibrio fischeri. Sci. Total Environ. 2014, 490, 807–814. [Google Scholar] [CrossRef]
  47. Corrales, J.; Kristofco, L.A.; Steele, W.B.; Yates, B.S.; Breed, C.S.; Williams, E.S.; Brooks, B.W. Global assessment of bisphenol A in the environment. Dose Response 2015, 13, 1559325815598308. [Google Scholar] [CrossRef]
  48. Ge, M.; Deng, W.; Wang, Z.; Weng, C.; Yang, Y. Effective decolorization and detoxification of single and mixed dyes with crude laccase preparation from a white-rot fungus strain Pleurotus eryngii. Molecules 2024, 29, 669. [Google Scholar] [CrossRef]
  49. Guo, Z.; Huang, A.; Gu, Z.; Guo, Z.; Yuan, L.; Gao, R.; Xin, Y.; Zhang, L. Ultrasound-assisted biomimetic mineralization immobilization improves the stability and catalytic performance of laccases derived from Bacillus licheniformis. Mol. Catal. 2025, 574, 114869. [Google Scholar] [CrossRef]
  50. Zdarta, J.; Jankowska, K.; Strybel, U.; Marczak, L.; Nguyen, L.N.; Oleskowicz-Popiel, P.; Jesionowski, T. Bioremoval of estrogens by laccase immobilized onto polyacrylonitrile/polyethersulfone material: Effect of inhibitors and mediators, process characterization and catalytic pathways determination. J. Hazard. Mater. 2022, 432, 128688. [Google Scholar] [CrossRef]
  51. Chmelová, D.; Ondrejovic, M.; Miertuš, S. Laccases as effective tools in the removal of pharmaceutical products from aquatic systems. Life 2024, 14, 230. [Google Scholar] [CrossRef] [PubMed]
Jox 15 00108 g001
Figure 1. FE-SEM image of synthesized Cu–PeLac protein–inorganic hybrids (A); elemental mapping for carbon (B), nitrogen (C), copper (D), and phosphorus (E); XRD pattern of Cu–PeLac hybrids (F); and CLSM images of FITC-labeled Cu–PeLac NFs under the green fluorescence channel (G) and bright-field view (H).
Figure 1. FE-SEM image of synthesized Cu–PeLac protein–inorganic hybrids (A); elemental mapping for carbon (B), nitrogen (C), copper (D), and phosphorus (E); XRD pattern of Cu–PeLac hybrids (F); and CLSM images of FITC-labeled Cu–PeLac NFs under the green fluorescence channel (G) and bright-field view (H).
Jox 15 00108 g001
Jox 15 00108 g002
Figure 2. Activity profiles of free and immobilized PeLac in the form of protein–inorganic hybrids NFs at various pH values (A) and temperatures (B).
Figure 2. Activity profiles of free and immobilized PeLac in the form of protein–inorganic hybrids NFs at various pH values (A) and temperatures (B).
Jox 15 00108 g002
Jox 15 00108 g003
Figure 3. (A) Storage stability of free and immobilized PeLac at 4 °C for 30 days. (B) Reusability of Cu–PeLac NFs and cumulative leaching over ten cycles.
Figure 3. (A) Storage stability of free and immobilized PeLac at 4 °C for 30 days. (B) Reusability of Cu–PeLac NFs and cumulative leaching over ten cycles.
Jox 15 00108 g003
Jox 15 00108 g004
Figure 4. Degradation of bisphenol A by free and immobilized PeLac: (A) degradation at various pH levels; (B) degradation at different bisphenol A concentrations; (C) repeated-batch degradation over multiple reuse cycles; and (D) degradation in the presence of known laccase inhibitors.
Figure 4. Degradation of bisphenol A by free and immobilized PeLac: (A) degradation at various pH levels; (B) degradation at different bisphenol A concentrations; (C) repeated-batch degradation over multiple reuse cycles; and (D) degradation in the presence of known laccase inhibitors.
Jox 15 00108 g004
Table 1. Immobilization of PeLac using Cu-based protein–inorganic hybrids at various protein concentrations.
Table 1. Immobilization of PeLac using Cu-based protein–inorganic hybrids at various protein concentrations.
Protein (mg/mL)Encapsulation Yield (%)Loading (mg/g of Synthesized Hybrids)Relative Activity (%) a
0.0584.7 ± 2.575.6 ± 5.6215 ± 16.6
0.1080.2 ± 2.797.8 ± 6.3278 ± 18.7
0.2578.8 ± 2.9163 ± 9.5333 ± 21.9
0.5054.5 ± 4.1191 ± 11.6205 ± 17.0
a Relative activity was calculated based on free PeLac activity, where 92.6 U/mg of total protein was considered 100%.
Table 2. Kinetic parameters and electrochemical properties of free and immobilized PeLac as protein–inorganic hybrids.
Table 2. Kinetic parameters and electrochemical properties of free and immobilized PeLac as protein–inorganic hybrids.
EnzymeVmax (µmol/min/mg Protein)Km (µM)Oxidation Current Peak (µA) a
Free PeLac93.1 ± 6.1118 ± 8.90.47 ± 0.03
Cu–PeLac NFs311 ± 19.348.6 ± 3.31.42 ± 0.07
a Measured at a potential of 0.5 V and a scan rate of 20 mV/s.
Table 3. Acute toxicity of Cu-NPs and Cu-based protein–inorganic hybrids NFs.
Table 3. Acute toxicity of Cu-NPs and Cu-based protein–inorganic hybrids NFs.
ParticlesEC50 Concentration (µg/mL)
Cu-NPs95.4 ± 6
Cu-NFs876 ± 48
Table 4. Thermal deactivation rate constant (kd) and half-life (t1/2) of free and immobilized PeLac as protein–inorganic hybrid NFs.
Table 4. Thermal deactivation rate constant (kd) and half-life (t1/2) of free and immobilized PeLac as protein–inorganic hybrid NFs.
Temperature (°C)Free PeLacCu–PeLac NFs
kd (h−1)t1/2 (h)kd (h−1)t1/2 (h)
400.05 ± 0.00313.2 ± 1.030.01 ± 0.00162.6 ± 3.2
450.10 ± 0.0076.93 ± 0.520.02 ± 0.00136.8 ± 1.9
500.25 ± 0.0152.78 ± 0.190.04 ± 0.00219.2 ± 1.1
550.66 ± 0.0281.05 ± 0.070.06 ± 0.00412.3 ± 0.8
601.05 ± 0.0490.66 ± 0.030.09 ± 0.0057.81 ± 0.6
652.23 ± 0.0880.31 ± 0.020.24 ± 0.0132.86 ± 0.3
704.08 ± 0.1230.17 ± 0.010.62 ± 0.0331.12 ± 0.2
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Patel, S.K.S.; Gupta, R.K.; Lee, J.-K. Immobilization of Pleurotus eryngii Laccase via a Protein–Inorganic Hybrid for Efficient Degradation of Bisphenol A as a Potent Xenobiotic. J. Xenobiot. 2025, 15, 108. https://doi.org/10.3390/jox15040108

AMA Style

Patel SKS, Gupta RK, Lee J-K. Immobilization of Pleurotus eryngii Laccase via a Protein–Inorganic Hybrid for Efficient Degradation of Bisphenol A as a Potent Xenobiotic. Journal of Xenobiotics. 2025; 15(4):108. https://doi.org/10.3390/jox15040108

Chicago/Turabian Style

Patel, Sanjay K. S., Rahul K. Gupta, and Jung-Kul Lee. 2025. "Immobilization of Pleurotus eryngii Laccase via a Protein–Inorganic Hybrid for Efficient Degradation of Bisphenol A as a Potent Xenobiotic" Journal of Xenobiotics 15, no. 4: 108. https://doi.org/10.3390/jox15040108

APA Style

Patel, S. K. S., Gupta, R. K., & Lee, J.-K. (2025). Immobilization of Pleurotus eryngii Laccase via a Protein–Inorganic Hybrid for Efficient Degradation of Bisphenol A as a Potent Xenobiotic. Journal of Xenobiotics, 15(4), 108. https://doi.org/10.3390/jox15040108

Article Metrics

Back to TopTop