Immobilization of Trametes versicolor Laccase by Interlinked Enzyme Aggregates with Improved pH Stability and Its Application in the Degradation of Bisphenol A

Abstract

Laccase from Trametes versicolor was immobilized via the formation of interlinking enzyme aggregates (CLEA). Its free and immobilized enzymes were characterized, and its efficiency was tested via the removal of bisphenol A (BPA) in aqueous solution. The resistances against thermal denaturation and pH variations were improved upon immobilization. Although the optimal pH of the enzyme was not modified by immobilization, the latter considerably increased its stability in the pH range of 2.0 to 8.0. The immobilized form was still 50% active after 6 months of storage, while the free form took 1 month to suffer a similar drop in activity. Both free and immobilized T. versicolor laccases were efficient in removing 200 µM BPA from aqueous solutions. The free laccase removed 79% and 92.9% of the compound during the first hour of reaction when 0.1 and 0.2 U were used, respectively. The immobilized form, on the other hand, removed 72% and 94.1% of 200 µM BPA during the first hour of reaction when 0.2 and 0.5 U were used, respectively. The immobilized enzyme allowed seven reuse cycles in the oxidation of ABTS and up to four cycles in the degradation of BPA. The results suggest that the laccase from T. versicolor may be useful in biological strategies aiming at degrading endocrine disruptors, such as BPA.

1. Introduction

Laccases (EC 1.10.3.2, benzenediol oxygen reductases) exhibit significant potential for use in various technological applications, attributable to their broad substrate specificity and high oxidizing capacity against both phenolic and non-phenolic compounds [1]. These enzymes have demonstrated a capability to degrade different recalcitrant xenobiotics, including dyes, pharmaceuticals, herbicides, pesticides and steroid estrogens [2,3,4]. Although the use of free laccases is quite advantageous energetically and environmentally, their application is often hindered by low operational stability and the inability to be reused, which ultimately results in high costs [2,5,6,7]. Additionally, the practical utilization of laccases is often hindered by several limitations, including instability in the presence of organic solvents, chemical additives, and pollutants, as well as challenges associated with enzyme recovery from reaction media for reuse [1,8]. These limitations arise primarily from the sensitivity of enzymatic activity to factors such as pH, temperature, and chemical conditions [9,10,11]. Part of this disadvantage can be overcome through enzymatic immobilization, a maneuver that significantly improves reuse and stability, both being features that allow process costs reductions of up to 50% [5,12]. Immobilization techniques have emerged as a viable strategy for preserving enzyme activity under extreme conditions. Given their ability to catalyze multiple reactions, identifying effective methods to enhance enzyme reusability is of considerable economic importance in biochemical processes. Stabilizing enzymes remains a critical approach to expanding their application in repetitive reaction cycles [13]. Several immobilization techniques have been applied to the immobilization of laccases [4,13,14]. Enzyme immobilization via the formation of interlinked (cross-linked) enzyme aggregates (CLEA) is a technique that does not need solid support, a feature that has been regarded as a crucial advantage. A solid support is not needed because interlinking is promoted by an appropriate agent (e.g., glutaraldehyde) when the carrier is co-precipitated with the enzyme. Additionally, CLEAs are known to be highly stable and resistant to denaturing agents [3,7].

With the exponential growth of industries, the number of pollutants produced and subsequently discarded has increased drastically. Several authors have investigated the use of laccases in free form or immobilized via different approaches, for the degradation of many pollutants [8,15,16,17]. Bisphenol A (BPA) is a chemical endocrine disruptor, widely used in the industrial production of epoxy resins, polycarbonate plastics, and other polymeric materials [18,19]. Due to its estrogen-like properties, BPA can accumulate in several organs and tissues, where it causes several adverse health effects. The latter include changes in epigenetic markers, disturbances in male and female reproduction caused by hyperplasia and hypertrophy in gonadotrophic cells, alterations in the transcription profile of genes coding reproductive signals, follicular atresia, and changes in hormone levels, among others [20,21]. It is thus important to remove or to degrade BPA as well as other phenolic pollutants from the environment. The most common methods for doing so, such as adsorption, distillation, chemical oxidation, extraction, and filtration, are often relatively ineffective in removing these pollutants from aqueous media [22]. The technique of enzymatic degradation, on the other hand, has been regarded as a promising approach, particularly with laccase, which uses molecular oxygen as the final electron acceptor [5,6]. Furthermore, the technique meets green chemistry standards, with the potential of degrading phenolic compounds, such as BPA, and strongly diminishing environmental impacts [14].

One of the most studied laccase producers is the white-rot fungus Trametes versicolor. It can be easily and economically cultivated through both submerged and solid-state fermentation using agriculture residues (e.g., wheat bran, sawdust, corn stover, sugar cane bagasse, etc.), producing elevated titles in laccase with the highest redox potential, a property that confers to this enzyme the ability to oxidize several phenolic and non-phenolic compounds [9,15]. One of the characteristics of the T. versicolor laccase is that the enzyme is particularly active within an acidic pH range, with the optimal pH varying depending on the substrate. For example, when ABTS (2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonate) is used as the substrate, the enzyme shows excellent activity at pH 2.0–3.0, but its stability is maximal in the pH range 4.0–6.0 [9,16]. Because of this, T. versicolor laccase can be used in long processes such as xenobiotic degradation only in the pH range 4.0–6.0. Several types of immobilization of T. versicolor laccase have been attempted in order to improve the stability of the enzyme and solve this problem of pH stability [11,23]. However, only a few studies have used the CLEA technique to immobilize this particular laccase [24]. Considering the above, the objective of this work was to immobilize the T. versicolor laccase via the formation of cross-linked enzyme aggregates. Efforts were made to evaluate the effects of the CLEA process on the physico-chemical and kinetics properties of the enzyme, as well on the capacity of immobilized laccase to remove bisphenol A from aqueous solutions. Furthermore, the experiments will be conducted in such a way as to allow a more clear and consolidated picture of the advantages and disadvantages of using the CLEA process as a technique for immobilizing the T. versicolor laccase.

2. Materials and Methods

2.1. Materials

The laccase substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as well as glutaraldehyde, bisphenol A, and Coomassie brilliant blue G-250 were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). The analytical-grade chemicals were of the highest purity available.

2.2. Microorganism and Production of Laccase

The Trametes versicolor used in this work belongs to the Basidiomycete Collection of the Laboratory of Biochemistry of Microorganisms of the State University of Maringá (UEM) and was registered in SISGEN under the number A4E5EC1 as of 1 November 2018. For the maintenance of fungus, it was cultivated on potato dextrose agar (PDA) plates. For the production of laccase, the fungus was cultivated in solid-state conditions, using 5 g of wheat bran as the substrate in 250 mL Erlenmeyer flasks. An initial humidity of 83% was achieved by adding a mineral solution [25] supplemented with 1% glucose and 0.1% yeast extract. After 7 days of cultivation at 28 °C in the dark, the crude extract was obtained by adding 15 mL of cold water to each flask, followed by shaking for 30 min at 10 °C, filtration through gauze, and centrifugation (10 min at 1792× g). A native sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) procedure was used, and this confirmed that a single 45 KDa-laccase was produced by the fungus, as described previously by another research group [9]. The crude laccase was lyophilized and stored at −20 °C until use.

2.3. Determination of Laccase Activity

Laccase activity was measured using 1.0 mM 2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS) as the substrate in 50 mM sodium acetate buffer (pH 5.0) at 40 °C (ε = 36,000 M⁻¹ cm⁻¹) for 5 min following the methodology previously described [26]. Enzymatic activity has been expressed in international units (U) (mol × 10⁻⁶·min⁻¹). Proteins were quantified by the traditional Bradford method [27]. The calibration curve was built with bovine-serum albumin. In the reuse experiments, the immobilized enzyme was washed with the same acetate buffer and submitted to a new round of determination of activity. To calculate the relative residual activity after each reuse, the absolute activity value yielded in the first use was taken as 100% [28].

2.4. Laccase Immobilization

Laccase immobilization was performed at 4 °C following a previously described methodology [29]. Ammonium sulfate was slowly added to the enzyme extract containing 0.35 g·mL⁻¹ of total proteins to achieve 55% (w/v) saturation. After the complete solubilization of ammonium sulfate, 150 mM of glutaraldehyde was used as the inter-linking agent. The suspension was maintained at 4 °C for 24 h and then centrifuged at 1792× g for 15 min. The precipitated materials (CLEAs) were washed four times in 50 mM sodium acetate buffer, pH 5.0, to remove excess ammonium sulfate and glutaraldehyde and stored at 4 °C in the same acetate buffer until use.

2.5. Evaluation of Immobilization Parameters

Two parameters were used to evaluate the efficiency of immobilization, immobilization yield (IY) and residual activity (RA), using the following equations:

$$\mathrm{Immobilization} \mathrm{yeld} (\%)={\displaystyle \frac{(\mathrm{UA}-\mathrm{UE})}{\mathrm{UA}}}\times 100$$

(1)

$$\mathrm{Residual} \mathrm{activity} (\%)={\displaystyle \frac{\mathrm{UH}}{(\mathrm{UA}-\mathrm{UE})}}\times 100$$

(2)

In Equations (1) and (2), UA represents the added units or units of activity offered for immobilization, UE is the output units or units of activity in the solution after the immobilization procedure, and UH is the immobilized units.

2.6. Physico-Chemical Properties

The oxidation rate of ABTS was used as the indicator reaction for characterizing the physico-chemical properties of both free and immobilized laccases. The standard incubation medium was a 50 mM sodium acetate buffer (pH 5.0), and the examined temperature range was 30 to 60 °C. The optimal pH conditions were identified at a temperature of 40 °C, utilizing the same substrate in a McIlvaine buffer solution, with pH values spanning from 2.0 to 8.0. The results were normalized to relative enzymatic activity, with the peak enzymatic activity set at 100%. The peaks of activity at 40 °C were 4.78 ± 0.50 and 1.84 ± 0.20 µmol/min·mg protein for free and immobilized enzymes, respectively. The peaks of activity at 50 °C were 6.84 ± 0.50 and 2.63 ± 0.30 µmol/min·mg protein for free and immobilized enzymes, respectively. In all experimental setups, inactivated enzymes served as negative controls. The thermal stability of the enzymes was evaluated by measuring residual activity after incubating both free and immobilized laccases in the absence of substrate at temperatures between 40 and 65 °C for up to one hour in a 50 mM sodium acetate buffer, pH 5.0. The pH stability was assessed by maintaining enzyme samples at various pH levels (2.0 to 8.0) within McIlvaine buffer for a duration of three hours. To assess long-term stability (storage stability), both free and immobilized enzymes were kept in a 50 mM sodium citrate buffer at pH 5.0 and stored in refrigeration (4–8 °C) for a duration of up to 6 months. For all stability assays, enzymatic activities were quantified using the ABTS assay system under standardized conditions (1.0 mM ABTS as the substrate in 50 mM sodium acetate buffer, pH 5.0, at 40 °C and 5 min of reactional time), as delineated in Section 2.3.

2.7. Determination of Kinetic Parameters

Reaction rates versus various ABTS concentrations (0.1–1.0 mM ABTS in 50 mM sodium acetate buffer, pH 5.0, at 40 °C and 5 min of reactional time) were measured, and the Michaelis–Menten equation,

$$\mathrm{v}={\displaystyle \frac{{\mathrm{V}}_{\max }[\mathrm{S}]}{{\mathrm{K}}_{\mathrm{M}}+[\mathrm{S}]}}$$

(3)

was fitted directly to the experimental data. In the equation, v is the initial reaction rate, [S] is the substrate concentration, K_M is the Michaelis–Menten constant and V_max is the maximal reaction rate. The non-linear least squares fitting procedure of Graph-Pad Prism Software 8.0 (GraphPad Software, Inc., Boston, MA, USA) was used.

2.8. Bisphenol A Degradation Measurement

The decomposition of BPA by both forms of laccase was monitored by quantifying the residual amounts of the compound using high-performance liquid chromatography. The incubation medium contained 50 mM sodium acetate buffer (pH 5.0), 200 µM BPA, and different quantities of free or immobilized laccase. The temperature was kept at 40 °C. The high-performance liquid chromatography was run in a C18 column (4.6 mm × 250 mm, 5 µm) at 40 °C with a flow rate of 0.8 mL/min. The elution medium was a mixture of methanol and water (70:30, v/v) and the detection was spectrophotometric (290 nm). Quantification was performed based on a calibration curve [29].

The residual BPA concentration versus time curves were fitted to the limiting exponential form of the integrated Michaelis–Menten equation,

$$\mathrm{C}={\mathrm{C}}_{\mathrm{o}}{\mathrm{e}}^{-({\mathrm{V}}_{\max }/{\mathrm{K}}_{\mathrm{M}})\mathrm{t}}={\mathrm{C}}_{\mathrm{o}}{\mathrm{e}}^{-({\mathrm{k}}_{\mathrm{o}\mathrm{b}\mathrm{s}})\mathrm{t}}$$

(4)

In Equation (4), C_o is the initial BPA concentration, C is the BPA concentration at time t and k_obs is the apparent first-order rate constant, which corresponds to the V_max/K_M ratio [30,31,32]. The fitting of Equation (4) to the experimental data was performed using the Scientist software 2.0 from MicroMath Scientific (Salt Lake City, UT, USA). The goodness of fit was judged by means of the model selection criterium [30] and the correlation coefficient. The time for half-maximal removal of BPA (t_½) was calculated as t_½ = 0.693/k_obs.

2.9. Statistical Analysis

All analyses were performed in triplicate. Data were expressed as mean ± standard deviation, and t-tests or ANOVA were conducted to assess significant differences between means. A 5% significance level (p ≤ 0.05) was adopted as the criterion of significance.

3. Results and Discussion

3.1. Evaluation of the Immobilization Efficiency of T. versicolor Laccase

The highest values for immobilization yield and activity retention were 100% and 78%, respectively, obtained after 24 h of reaction with a final concentration of 150 mM of glutaraldehyde. The technique used for the immobilization of T. versicolor proved to be quite efficient. Previous studies have reported the efficiency of the CLEA immobilization process for: (1) the laccase of Oudemansiella canarii, where the highest immobilization yield and activity retention values were 95% and 78%, respectively [28]; (2) a T. versicolor laccase, where the immobilization efficiency was 48% for CLEAs, with a recovery of 32% [33]; and (3) a laccase from Aureobasidium pullulans, achieving a recovery of about 80% to 65%, with an immobilization yield of 60% [3].

3.2. Activity and Stability as a Function of Temperature

Figure 1 and Figure 2 depict the influence of temperature on the activity and stability of both forms of enzymes. Figure 1 shows that despite the lower activity of the immobilized form, the temperature dependence was not significantly modified. Both the free and immobilized forms were more active at 50 °C. Figure 2 shows the residual activities of free and immobilized enzymes after exposure to different temperatures, between 40 and 65 °C, in the absence of substrate for up to 60 min. The activities of both enzyme forms in Figure 2 are expressed as percentages of the activities that were measured at the optimal temperature of 50 °C. The thermal stability of the laccase was improved by immobilization. On exposure to the temperatures of 40, 50, and 60 °C, both the free and the immobilized forms demonstrated high stability for 60 min. The half-life (t_½) of the free enzyme was 28 min at 65 °C, while for the immobilized enzyme it was greater than 60 min. This behavior is similar to that observed for the laccase of O. canarii immobilized by the same methodology [28].

It is also similar to that of the Pleurotus ostreatus laccase immobilized on MANAE-agarose, in which ionic adsorption is the underlying immobilization phenomenon [4]. An important criterion when it comes to judging the applicability of an immobilized laccase is its thermal stability, which is expected to be as high as possible. The binding of the enzyme to a matrix or support makes it more resistant to heat denaturation. This improvement in stability may be due to an increased structural stability or to a decreased exposure to denaturing agents [4]. The multiple binding networks in the protein structure and interactions between Cu²⁺ molecules with copper centers may influence the thermal activity and stability of the laccases [34].

3.3. Effects of pH

Between pH 2 and 7, both enzymes experienced almost linear declines in their activities, as revealed by Figure 3A,B (). The activity of both enzyme forms was, thus, restricted to the acidic range; at pH 7, both were practically inactive. Figure 3A,B also shows the stability of pH (), which was measured via the remaining activity at pH 5.0 when the enzymes were incubated at different pH values (2.0–8.0) for 3 h. The immobilized laccase was quite stable in the pH range of 2.0–8.0 (Figure 3B), whereas the stability of the free enzyme was restricted to a pH range of 4.0–6.0 (Figure 3A). Although the optimal pH for both free and immobilized fractions is in the more acidic range (2.0 and 3.0), T. versicolor laccase is more stable at a pH of 4.0 to 6.0, which justifies its use in this pH range. This distinct stability at different pH levels may occur due to substrate specificity, the redox potential difference between the enzyme and substrate, and the availability of oxygen [34]. Fungal laccases typically have an optimal pH in the acidic range, and pH stability can vary widely depending on the source of the enzyme [9,10,35]. Applying our data to these notions, it seems appropriate to conclude that the CLEA method for immobilizing T. versicolor laccase improved operational stability, increasing thermal stability but mainly increasing pH stability, without altering the optimal temperature and pH for activity. Other studies have reported that the thermal and pH properties, both in the free form and in the immobilized form of laccase using different immobilization methodologies, yielded results similar to those reported in this work, as can be seen in

Table 1. Comparison of thermal and pH properties of free and immobilized laccase in different matrices.

Microorganism/Immobilization Matrix	Properties	Reference
Trametes versicolor CLEA	Highest activity between 50 and 55 °C. Immobilized laccase was more stable in the regions of 60 and 65 °C. Optimal pH in the more acidic range (2.0 and 3.0) and greater stability in the range of 4.0 to 6.0. Thermal and pH stability were improved with immobilization.	This work
Trametes versicolor NaY Zeolite	Optimal pH at 4.0 (free) and 6.0 (immobilized). Greater stability at pH 7.0 after immobilization. Maximum activity at 30 °C (free) and 50 °C (immobilized).	[38]
Trametes versicolor Chitosan/halloysite	Optimal pH at 6.0 (free) and 5.0 (immobilized). Immobilized laccase more stable under acidic conditions. Maximum activity at 45 °C (free) and 35 °C (immobilized). The activity of the immobilized enzyme was greater than that of the free enzyme at temperatures, different from 45 °C.	[39]
Ganoderma lucidum MANAE-agarose	Optimal activity at pH 5.0 (both free and immobilized). Reduced enzyme sensitivity to alkaline pH after immobilization. Maximum activity at 60–65 °C. Greater stability at 40 and 55 °C after immobilization.	[29]
Mono and co-culture Trametes villosa/Pycnoporus sanguineus CLEA	Best catalytic activity between 50 and 60 °C. Greater thermal stability after immobilization, maintaining 100% activity after 180 min, while the free form had less than 90%. The optimal pH range for laccase activity in the free form was between 2.4 and 2.6, and 2.6 and 3.6 for the immobilized form.	[40]
Brevibacterium halotolerans N11 (KY883983) Alginate-gelatin	The optimal pH values were 5.0 (free) and 6.0 (immobilized). The immobilized laccase was stable at pH values ranging from 5 to 7 compared to the free enzyme. The optimal temperature of the immobilized laccase increased from 35 °C to 40 °C. The immobilized laccase was stable at 58 °C.	[41]

. Increased thermal stability in immobilized enzymes has been interpreted as a consequence of the limitation of the thermal movement, which leads to denaturation at higher temperatures [36]. With respect to the increased pH stability, one has to take into account that the pH has a pronounced influence on the conformation of proteins in general. On this issue, it has been pointed out that immobilization reduces the conformational flexibility, thus diminishing the probability of the enzyme assuming conformations that result in diminished catalytic activities [37].

3.4. Kinetics

Figure 4 shows the substrate saturation curves for ABTS obtained with the free and immobilized laccases. Both enzyme forms obeyed the Michaelis–Menten equation. The kinetic constants, K_M and V_max, were 0.09 ± 0.02 mM and 5.11 ± 0.24 mmol min⁻¹ (mg protein)⁻¹ for the free enzyme and 1.061 ± 0.62 mM and 4.94 ± 1.324 mmol min⁻¹ (mg protein)⁻¹ for the immobilized enzyme, respectively. The immobilization of laccase caused a significant increase (p ≤ 0.05) in the K_M value and a slight and non-significant reduction (p > 0.05) in V_max, resulting in a decrease in the V_max/K_M ratio from 56.78 (free enzyme) to 4.89 (immobilized enzyme). This means a decrease in catalytic efficiency of 100 × (1 − 4.89/56.78) = 91.3% at low substrate concentrations when ABTS was the substrate. This may be the result of several factors. Conformational changes in the enzyme molecule may occur due to alterations in the tertiary structure of the active site. Modified stereochemical interactions may be induced by the altered conformation. Finally, after the immobilization step, the microenvironment surrounding the catalytic site may also have been modified, which especially affects the electrostatic interactions between the catalytic groups and the substrate. The microenvironment close to the enzyme differs from that of the reaction solution, and the effects of diffusion or mass transfer arise from the resistance to substrate diffusion to the enzyme’s catalytic site and the product to the solution. On the other hand, the very pronounced decrease in catalytic efficiency found with ABTS upon immobilization may not be necessarily valid for all substrates, a question that will be investigated further using the environmental contaminant bisphenol A [3,18,42,43].

3.5. Reusability of the Immobilized Laccase in the Oxidation of ABTS

Reusability and long-term storage are two of the most important advantages of immobilized enzymes, which lead to lower costs and feasible large-scale applications [28]. In the present work, consecutive cycles of the ABTS oxidation procedure were employed to evaluate the immobilized laccase reusability. As revealed by Figure 5, even after 10 cycles, the immobilized enzyme still possessed 50% of its original activity. The slight decrease in oxidation efficiency in the course of the cycles may be due to the gradual release of the enzyme during the operations, a consequence of the weak interactions between the support and enzyme. In a similar work, a laccase immobilized onto magnetically modified biochar derived from apple branches maintained above 50% of its relative activity until the seventh recycling of ABTS oxidation [44].

Long-term stability is expected to be increased by immobilization. In the present work, this question was approached by periodically checking the activities of samples of both enzyme forms, which had been stored in a refrigerator (4–8 °C). The immobilized enzyme still presented more than 50% of its initial activity after 6 months of storage. The free laccase, on the other hand, saw its activity drop to slightly less than 50% after just 1 month. There is thus a clear superiority on the part of the immobilized enzyme with respect to long-term stability.

3.6. Bisphenol A Degradation

Experiments were also conducted in which the removal of 200 μM bisphenol A was tested. These experiments had two main purposes. The first one was to test if the CLEA-immobilized laccase of Trametes versicolor can remove an important and deleterious contaminant such as bisphenol A [3,18,42,43]. The second purpose was to find out if the ratio of removal of bisphenol A at low concentrations by the immobilized and free enzyme forms was as unfavorable as observed for the transformation of ABTS. The results of these experiments are shown in Figure 6 and

Table 2. Parameters obtained by fitting Equation (4) to the experimental bisphenol A degradation curves obtained with both free and immobilized laccases. The equation was fitted to the experimental curves shown in Figure 6 by means of a non-linear least squares procedure, as described in Section 2.

Enzyme Form	Units	kobs (min−1)	t½ (min)	Model Selection Criterion	Correlation Coefficient
Free	0.1	0.0217 ± 0.0005	31.93 ± 0.03	5.733	0.999
	0.2	0.0379 ± 0.0013	18.28 ± 0.07	5.568	0.998
	0.3	0.0512 ± 0.0032	13.51 ± 0.84	4.934	0.998
Immobilized	0.2	0.0125 ± 0.0006	55.17 ± 2.70	3.871	0.995
	0.5	0.0473 ± 0.0038	14.64 ± 1.17	4.303	0.996

. Figure 6A shows the removal curves obtained with the free enzyme, and Figure 6B shows the corresponding curves obtained with the immobilized laccase. The experimental curves were analyzed by means of Equation (4), which is the limiting exponential form of the integrated Michaelis–Menten equation, and is valid when the substrate concentrations are lower than the K_M or at least close to the K_M. This equation was fitted to the experimental curves, allowing the determination of the apparent first-order rate constant (k_obs), which is a function of enzyme concentration and corresponds to the V_max/K_M ratio. The time for half-maximal removal can be calculated directly from this parameter. The continuous lines in Figure 6 were calculated using the optimized values of the rate constant (k_obs ≈ V_max/K_M). Comparison of these lines with the experimental points reveals relatively good agreement in all cases. This is also indicated by the relatively high values of the model selection criterium parameter and of the correlation coefficient, listed in Table 2. All this indicates that the estimated k_obs values are a good estimate for the V_max/K_M ratio. The estimation of k_obs has the additional advantage that it allows the determination of the time for half-maximal removal (t_½). Values of the rate constants and the corresponding times for half-maximal removal are also listed in Table 2, in addition to the amounts of enzyme that were used, expressed as enzyme units, determined using the reaction rates with saturating ABTS concentrations. Close analysis of the data in Table 2 reveals that similar removal rate constants of bisphenol A by the free and immobilized laccases were found when 0.3 and 0.5 units, respectively, were used. A comparative calculation, similar to that employed for assessing the ABTS transformation capacities of the free and immobilized laccase, can be performed for the bisphenol A transformation using the V_max/K_M ratios in Table 2. Using the data in which the degradation was measured with 2 units, one arrives at an efficiency loss of 100 × (1 − 0.0125/0.0379) = 67.02%. As described above, the transformation of ABTS at low concentrations was 91.3% affected by immobilization, ten times more enzyme thus being required for similar transformations. The transformation of bisphenol A is thus less strongly affected by immobilization, and only three times more enzyme is required for similar transformations. Additionally, when using 0.5 U of the immobilized laccase, the system could degrade 80% of 200 µM BPA in the fourth cycle of reuse.

The necessity of using more enzyme for degrading BPA with the immobilized form may be explained roughly in the same terms as in the case of ABTS (see Section 3.4). Nevertheless, Figure 6 reveals that the complete degradation of bisphenol A can be achieved in a reasonable time, and the need for more immobilized enzyme is compensated for by its stability to pH variations and the possibility of reusing it several times. Finally, in

Table 3. Comparison of studies on transformation and degradation of bisphenol A using immobilized laccase from T. versicolor.

Type of Immobilization	Main Results	Reference
Cross-linked aggregates	72% and 94.1% of 200 µM BPA were removed after 1 h of treatment at 40 °C and pH 5.0 using 0.2 and 0.5 U of immobilized laccase, respectively. The immobilized enzyme allowed 7 reuse cycles in the oxidation of ABTS and up to 4 cycles in the degradation of BPA.	This work
Glutaraldehyde cross-linked chitosan beads	Degradation of 20 mg/L (87.6 µM) BPA in 2 h. The immobilized enzyme retained 71.24% of its original activity after 10 repeated catalytic cycles.	[7]
Multi-channel ceramic membrane	Degradation of 20 mg/L (87.6 µM) BPA in 24 h. The enzyme showed a degradation rate of 79.0 ± 0.1 μmol/min/U.	[45]
Ba-alginate beads	Maximum BPA degradation of 84.34% was obtained at a temperature of 40 °C after 50 min using an initial BPA concentration of 2 mg/L (8.76 µM).	[42]
Cu-alginate beads	0.5 g immobilized enzyme was able to degrade 96.12% of BPA (10 mg/L, 43.8 µM) at pH 5.0, 30 °C, 150 rpm and 23 h of reaction	[43]

, we present a comparison of the immobilized laccase from Trametes versicolor in this work and that yielded by other immobilization methods found in the literature. The comparison allows us to conclude that the enzyme immobilized here is more efficient in degrading BPA than the previously reported preparations.

4. Conclusions

Based on the results that were achieved, the immobilization of the laccase from Trametes versicolor by means of the CLEA technique can be considered a successful undertaking. An equally successful one involves the observation that the immobilized enzyme still possessed half of its capacity for oxidizing ABTS over more than 10 cycles of reuse. Both thermal and pH stabilities of the immobilized laccase were clearly superior to those of the free enzyme. When tested for their capacity for removing a true pollutant of the environment, namely, BPA, both enzyme forms proved to be efficient. In this particular respect, the superiority of the immobilized enzyme resides in its stockpile stability and reuse possibilities. Taking all observations into account, finally, it can be concluded that T. versicolor laccase presents favorable perspectives in relation to being useful in biological strategies aiming at degrading endocrine disruptors such as BPA and other pollutants.