Laccase Immobilization on Carbon-Based Materials Derived from Spent Brewery Grains: Optimization and Stability Evaluation

TL;DR

This study explores using carbon materials from brewery waste to immobilize laccase, finding a greener option with good stability for environmental applications.

Contribution

A sustainable, chemical-free method for laccase immobilization using biochar from spent brewery grains is proposed and evaluated.

Findings

SBG-AC achieved 80% laccase immobilization, while SBG-BC required no chemical activation and retained ~60% activity after 24 hours.

SBG-BC showed lower substrate affinity but higher stability compared to free laccase.

The study highlights the potential of SBG-BC for wastewater treatment and environmental monitoring.

Abstract

Enzyme immobilization onto solid supports enhances their stability, reusability, and efficiency. This work investigates the physical immobilization of laccase (Lac) from Trametes versicolor (purchased, EC 1.10.3.2, ≥0.5 U/mg) onto two carbon-based materials: activated carbon (AC) and biochar (BC), obtained from spent brewery grains (SBGs) through microwave pyrolysis (with and without chemical activation, respectively), generating SBG-AC/Lac and SBG-BC/Lac. Various immobilization conditions (pH 3.5–6.5, Lac concentration 1–10 mg/mL) were tested, with immobilization up to 80 ± 6% (for Lac 1 mg/mL, pH 5.0 in SBG-AC/Lac) and maximum activities of 5.5 ± 0.2 U/g (SBG-AC/Lac) and 4.6 ± 0.5 U/g (SBG-BC/Lac) at pH 3.5 and 40 °C. Although SBG-AC led to a higher immobilization %, SBG-BC was a greener alternative, requiring no chemical activation during production. Kinetics analysis with a typical Lac chromogenic substrate revealed higher values of KM (Michaelis constant) for SBG-BC/Lac compared with free Lac (Lacf) (indicating lower substrate affinity), but higher stability, retaining ~60% activity after 24 h, while Lacf was nearly inactive. These results demonstrate the potential of SBG-BC as a sustainable support for Lac immobilization in applications such as wastewater treatment and environmental monitoring.

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Full text

1. Introduction

Enzymes are proteins that accelerate biochemical reactions by lowering the activation energy, acting as biological catalysts. They can facilitate several reactions with high specificity, selectivity, and evidence of activity, generally under mild reaction conditions [1]. By reducing the number of synthesis steps and solvent use, enzymes enable catalytic processes that are both cost-efficient and environmentally sustainable [2]. For these reasons, the use of enzymes in industrial processes, such as food production [3], cosmetics [4], textiles [5], and even in wastewater treatment [6], has increased over the years. Free enzymes belonging to the class of oxidoreductases, such as lignin peroxidase, manganese peroxidase, versatile peroxidase, and laccase (Lac) are among the most used, for example, in wastewater treatment [7]. Among them, Lac (EC 1.10.3.2, benzenediol:oxygen oxidoreductase) is a member of the multicopper enzyme family that catalyzes the oxidation of hydrogen-donating compounds with a structure similar to p-polyphenol, concurrently reducing oxygen to water [8]. These enzymes can be obtained from plants, insects, and fungi, with laccases from the fungus Trametes versicolor and the Japanese lacquer tree Rhus vernicifera being among the best-characterized. In fungi, Lac plays a role in spore pigment formation, phenolic compound detoxification, and lignin degradation [9]. Due to its ability to catalyze the oxidation of a wide variety of aromatic substrates, it has been used—either as a purified enzyme or in crude enzymatic extracts—for the degradation of contaminants present in wastewater, including pharmaceutical drugs [7], pesticides [10], and dyes [11]. However, as reviewed by Chen et al. [12], enzymes are proteins that might be denatured under harsh conditions of temperature, pH, or use of organic solvents, which, together with high production costs, limit their widespread industrial application. To address these issues, enzyme immobilization on solid supports has emerged as an effective solution [2,13]. While incurring additional costs, immobilizing enzymes facilitates post-process recovery, improves stability under extreme conditions, and enables reuse, thereby enhancing control over catalysis [2,7]. Consequently, immobilization makes enzymes particularly suitable for continuous operations, along with the use of multi-reaction enzyme systems [14]. Immobilization typically involves attaching enzymes to insoluble carriers using techniques such as entrapment, covalent bonding, cross-linking, and adsorption, as reviewed by Nguyen et al. [15]. Among the existing Lac immobilization methods, the simplest is adsorption, which involves enzymes in solution being bound to the support through weak, nonspecific forces (e.g., van der Waals interactions, hydrogen bonding, ionic interactions) [16]. As immobilization by adsorption does not involve functionalization, it is generally considered a non-destructive process for enzymes. Supports for enzyme immobilization include organic materials like cellulose and chitosan [17], inorganic substances such as silica [18] and zeolites [19], and metal–organic frameworks [20]. However, the high production costs of these materials and their limited scalability reinforce the need to develop innovative materials and techniques to overcome these limitations, as reviewed by Patti et al. [21]. In this sense, agro-industrial wastes have emerged as an alternative, contributing to sustainable resource management given their annual production of millions of metric tons [22]. Lignocellulosic wastes are mainly generated by agricultural industry activities, and are composed of cellulose, hemicellulose, and lignin, with varying proportions depending on the source. Due to the presence of functional groups such as carboxyl, hydroxyl, and amino, these wastes have been used as supports for enzyme immobilization, either in their raw or carbonized form [23]. In fact, the production of carbon-based materials such as biochar (BC) or activated carbon (AC) using the referred wastes also offers advantages such as their inert nature and high surface area, developed pore structure, rich surface oxygen-containing functional groups, good dispersibility, and biocompatibility for stable and high-load enzyme immobilization [14,24,25,26,27,28].

This work aims to study the physical immobilization of the oxidoreductase Lac onto two carbon-based materials with distinct adsorptive properties, AC and BC, derived from spent brewery grains (SBGs), a lignocellulosic waste which results from the brewing process. SBG accounts for approximately 85% of the total by-products generated in the brewing industry, amounting to an estimated 3.4 million tons annually in the European Union [29]. The utilization of SBGs as a carrier for Lac immobilization has already been explored to promote the valorization of this waste (e.g., [30]). In addition, the production of AC and BC for wastewater treatment using SBGs as raw material (SBG-AC and SBG-BC, respectively) has been assessed as a sustainable alternative for managing this type of waste [31,32]. However, to the best of our knowledge, SBG-based carbon adsorbents have not been explored for enzyme immobilization. This study will evaluate such immobilization in terms of enhanced enzyme stability and catalytic efficiency compared to the free enzyme. By combining the adsorption properties of support materials with the catalytic efficiency of enzymes, the resulting composite materials might offer improved performance that can benefit industrial processes, such as wastewater treatment. In addition, by repurposing agro-industrial waste as SBGs, this work contributes to sustainable resource management and aligns with the EU’s Circular Economy Action Plan [33].

2. Results and Discussion

Several studies in the literature on the immobilization of Lac showed that the binding capacity of Lac onto support materials is influenced by the materials’ textural and chemical properties (e.g., chemical composition, surface area), functionalization, and reaction environment [15]. In this work, Lac immobilization was achieved through adsorption onto two distinct materials, SBG-AC and SBG-BC, without additional functionalization. Previously obtained results from X-ray photoelectron spectroscopy (XPS) characterizing SBG-AC [34] and SBG-BC [32] revealed four main peaks corresponding to C1s, O1s, N1s, and Si2p levels. The presence of oxygen- and nitrogen-containing functional groups, such as carboxylic (–COOH or –COOR) and carbonyl groups (–C=O group in quinones), and pyridinic structures suggests potential interaction with enzymes, both affecting adsorption and/or catalysis. In addition, it is important to consider the point of zero charge (PZC) of the two support materials and the enzyme’s isoelectric point, as they influence the immobilization process. The PZC of SBG-AC and SBG-BC were determined to be 5.0 [35] and 5.6 [34], respectively. These parameters can influence the physical immobilization of the enzyme onto the support, and can also determine its stability under operational conditions, as discussed below.

2.1. Characterization of Carbon-Based Materials

The textural parameters of SBG-AC and SBG-BC were evaluated using nitrogen adsorption isotherms and are presented in Table 1. SBG-AC showed a higher specific surface area (SBET) and total pore volume (Vp) than SBG-BC. The difference in these parameters is due to the chemical activation of SBG-AC with potassium carbonate, which promotes high volatile release and the development of a porous structure. In accordance, SBG-BC and SBG-AC were characterized in previous works regarding SBET and pore volume, with 1405 m^2^/g and 0.67 cm^3^/g, respectively, for SBG-AC [31], and 312 m^2^/g and 0.14 cm^3^/g for SBG-BC [32]. These values are comparable to, or higher than, those of commercial carbon materials, showing that, together with the lower production costs associated with waste repurposing and microwave pyrolysis to reduce energy consumption, these materials are competitive alternatives for further modification to be used in environmental applications.

The physical immobilization of Lac onto SBG-AC and SBG-BC proceeds via an adsorption process onto the porous carbon materials, which can be described as a sequence of steps. Initially, Lac molecules diffuse from the bulk solution to the external surface of the carbon particles. At this stage, enzyme attachment might primarily be governed by electrostatic attraction or repulsion between the enzyme’s surface charge and the charged functional groups on the carbon materials, as discussed in Section 2.2. As previously reported, X-ray photoelectron spectroscopy (XPS) analyses of the studied carbon materials [32,34] revealed the presence of oxygen- (e.g., –COOH and –OH) and nitrogen-containing functional groups, which promote interactions with the enzyme and influence its adsorption behavior. This hypothesis is supported by the work of Imam et al. [24] and Naghi et al. [36], who further revealed the important role of carboxylic groups on the carbon materials in enhancing enzyme anchorage, thereby increasing immobilization efficiency. These interactions are strongly affected by the protonation and deprotonation states of surface functional groups, which vary with pH. In addition to electrostatic interactions, hydrogen bonding and π–π interactions between the aromatic domains of the carbon matrix and aromatic amino acid residues of the enzyme also contribute to Lac adsorption, as demonstrated by Pandey et al. [25].

After the initial attachment, Lac molecules can diffuse along the surface and into accessible meso- and macropores, where additional stabilization occurs through a combination of hydrogen bonding (between polar amino acid residues and oxygen-containing groups such as –COOH and –OH) and hydrophobic/π–π interactions with aromatic domains of the carbon matrix. As adsorption progresses, the available surface becomes increasingly occupied, eventually approaching near-monolayer coverage, in which enzyme–enzyme contacts and steric effects may limit further binding and can slightly restrict substrate diffusion to the active sites. The higher SBET and more developed pore network of SBG-AC (Table 1) provide a larger accessible surface and more adsorption sites for Lac, which is consistent with the higher immobilization percentage obtained for this material, whereas the less developed porosity of SBG-BC offers fewer accessible sites and leads to lower enzyme loading (see Section 2.3). This was also observed by Li et al. [37], who, when evaluating the SBET of two carbon materials—maple biochar and spruce biochar—found differences in immobilization potential, with more Lac immobilized on the biochar with a higher SBET and pore volume (maple biochar).

2.2. Effect of pH on Immobilization

The immobilization percentage (%) of Lac onto SBG-AC and SBG-BC varied between 15 ± 5% and 80 ± 6%, depending on the concentration of Lac in immobilization suspensions (1.0 to 10.0 mg/mL) and the immobilization pH (3.5, 5.0, and 6.5). Figure 1 shows the results of the effect of pH on the Lac immobilization % on SBG-AC and SBG-BC (Figure 1A) as well as the enzymatic activity (U/g DW) of the resulting composites (Figure 1B), which were obtained at a fixed concentration of Lac (1.0 mg/mL) in the immobilization suspension.

According to Figure 1A, SBG-AC/Lac demonstrated a higher immobilization % of Lac (80 ± 6%) at pH 5.0, compared with the remaining immobilization pH (3.5 and 6.5). It must be considered that, at the overall best pH of immobilization (pH 5.0), SBG-AC (PZC = 5.0) has a neutral surface net charge. Considering an isoelectric point of 2.8 for free Lac (from T. versicolor) [38], at pH 5.0, electrostatic repulsion is not expected to occur between SBG-AC and Lac. Nevertheless, at pH 6.5, it is possible that the likely prevalence of negative surface charges resulted in the repulsion between the enzyme and the support, alongside an accentuated decrease in the immobilization percentage (Figure 1A). In contrast, SBG-BC/Lac exhibited a higher % of Lac immobilization at the highest pH tested (6.5), with a maximum value of 58 ± 6%. At pH 6.5, both SBG-BC (PZC = 5.6) and Lac were mainly negatively charged, so it was expected that repulsion between the material and the enzyme would occur, as observed for SBG-AC/Lac at a similar pH. Therefore, the obtained results seem to suggest that the success of immobilization of Lac in those materials is not completely ruled by electrostatic interactions. In addition, although generally most fungal laccases have acidic isoelectric points, values can vary from 2.8 to 4.6, depending on the basidiomycete species [38,39]. Conversely to the observed results for SBG-BC/Lac, Imam et al. [24] showed that the best immobilization of Lac (65.6%) occurred at pH 3.1, where the negatively charged surface of the rice straw BC was suitable for ionic interaction with Lac (from fungal strain T. maxima IIPLC-32) that had an overall positive charge. The authors observed a reduction in the immobilization yield with increasing pH, due to electrostatic repulsion between the negatively charged support material and the enzyme (isoelectric point of 5). Similarly, Silva et al. [40] observed an increase in the loading of Lac (from T. versicolor) in an avocado seed-based BC at pH 4.0, but referred to the contribution of the ionic interaction and covalent binding due to functionalization with glutaraldehyde. The authors verified that, at pH 4.0, the ionic interaction among the materials and the enzyme was maximized as the PZC of the materials tested was 5.7 and 5.4, being positively charged, while the enzyme was negatively charged.

Considering the activity of immobilized Lac (Lac_i_) in the composites (Figure 1B), similarly to the previous results on immobilization %, a slightly higher activity (3.5 ± 0.1 U/g DW) in SBG-AC/Lac was observed at pH 5.0, compared with the remaining immobilization pH, although the values did not differ statistically. Similarly, the different immobilization pH for SBG-BC/Lac did not significantly affect the activity of the composites. This is in accordance with the results of Li et al. [37], who found no significant differences comparing the recovered activity of Lac (from Coprinus comatus) in two BC composites, despite the differences in the % of immobilization.

2.3. Effect of Laccase Concentration on Immobilization

At the overall best pH of immobilization (considered to be pH 5.0, according to results presented in Figure 1), the effect of enzyme concentration (1.0, 5.0, and 10.0 mg/mL) on the immobilization % onto SBG-AC and SBG-BC was evaluated (Figure 2).

The results in Figure 2 indicated a decrease in the immobilization % with increasing Lac concentration for SBG-AC/Lac. In contrast, SBG-BC/Lac showed the opposite trend, with a higher immobilization % at higher Lac concentrations (Figure 2A). Specifically, the immobilization % for SBG-AC/Lac was 80 ± 6, 79 ± 7, and 73 ± 5%, respectively, for the increasing Lac concentration (1.0, 5.0, and 10.0 mg/mL), which corresponds to 80 ± 6, 396 ± 33, and 727 ± 52 mg of Lac immobilized in each g of SBG-AC. For SBG-BC/Lac, enzyme loading at pH 5.0 was 53 ± 6, 73 ± 6, and 64 ± 5%, corresponding to 54 ± 6, 368 ± 32, and 634 ± 50 mg of Lac immobilized in each g of SBG-BC. Also, at the same immobilization suspension concentration, it was found that, in general, SBG-AC/Lac presented a higher immobilization % of Lac than SBG-BC/Lac. The immobilization % obtained in this work is comparable to or higher than the values for carbon-based materials derived from agricultural wastes, which typically range from ~35–70% depending on the immobilization conditions and the textural and surface characteristics of the material [16,26,27,37,40], demonstrating the suitability of SBG-derived materials for high-capacity Lac immobilization. Specifically, Al-sareji et al. [27] observed the highest yield (65.2%) of Lac (from T. versicolor) immobilization in AC from coconut shell, at a concentration of 2 mg/mL (at pH 5.0). The authors also observed that above a Lac concentration of 2 mg/mL, the system reached an equilibrium due to a decrease in the available surface sites on AC. The occupation of the available pores on the carbon-based materials’ surface caused by increasing enzyme dosage was also reported by Pandey et al. [25]. Consistently, the results presented here demonstrate that at Lac concentrations above 5 mg/mL, immobilization does not increase proportionally on either AC or BC supports, indicating saturation of the support materials. In addition, as previously reported, pH is not the only factor influencing Lac immobilization; the surface area and pore volume of the supports can also affect it. The highest % of Lac immobilization on AC composites could be related to the surface area of the material, with SBG-AC presenting higher values, as previously reported (see Section 2.1), than SBG-BC. The increased surface area of SBG-AC offers more sites for enzyme attachment, enabling multi-point interactions between the protein and the support material, in agreement with the results reported by Li et al. [37]. These authors observed a higher immobilization yield (64.2%) of Lac (from C. comatus) at a concentration of 16 mg/mL (at pH 3.0), with 11.1 mg of Lac per g of maple BC (surface area 613 m^2^/g and pore volume 0.69 cm^3^/g), compared with spruce BC, which showed 37.6% of immobilization yield, with 7.6 mg of Lac per g of support (surface area 86.3 m^2^/g and pore volume 0.065 cm^3^/g).

Considering the enzymatic activity of the obtained composites under the conditions described (Figure 2B), it was observed that, for both SBG-AC/Lac and SBG-BC/Lac, the activity increased with an increase in concentration of Lac in the immobilization suspension, reaching a maximum at the highest Lac concentration (10.0 mg/mL), namely 5.5 ± 0.2 and 4.6 ± 0.5 U/g for SBG-AC/Lac and SBG-BC/Lac, respectively. However, for both composite types, statistically significant differences were observed only between the two lower concentrations (1.0 and 5.0 mg/mL). Using AC fibers as a support material, Zhang et al. [41] observed a similar trend when immobilizing increasing concentrations (0.5–5.0 mg/mL at pH 5.0) of Lac (from Aspergillus). These authors determined that the maximum activity of Lac_i_ was 97.1 ± 2.5 U/g at a Lac concentration of 3 mg/mL in the immobilization solution, declining as the Lac concentration increased. This response was attributed to the increased intermolecular steric hindrance exerted by higher Lac concentrations, which restrained the diffusion of substrates and products. In accordance, Fortes et al. [42] reported a decrease in the activity of Lac recovered after immobilization on functionalized magnetic particles with an increase in Lac dosage, justified by saturation of the support by the enzyme, restraining the dispersion of substrate and product.

2.4. Stability of Free and Immobilized Laccase

2.4.1. Effect of Operation pH on Enzyme Activity

Enzyme stability across different operation pH levels is an important parameter influencing its efficacy in support materials, as pH can affect the ionization state of the amino acids that compose enzymes and consequently alter their structure and activity [43]. In addition, it can influence the adsorption of the enzyme on the support, promoting its desorption or leaching. These changes can result in reduced catalytic activity of immobilized enzymes, reduced reusability, and overall lower stability, especially in continuous or long-term operations. In this work, the impact of operation pH on the activities of SBG-AC/Lac, SBG-BC/Lac, and Lac_f_ was evaluated. Figure 3 shows the results of the enzymatic activity of the composites at different operation pHs (3.5 to 6.5) in the reaction media (Figure 3A) and the relative activity (%) of SBG-BC/Lac compared with Lac_f_ (Figure 3B) under the same conditions.

For both composites, the largest activity was found at pH 3.5, substantially decreasing with the increase in pH in the reaction media and presenting residual activity at the highest pH tested (pH 6.5) (Figure 3A). Imam et al. [24] observed similar results for Lac (from fungal strain Trametes maxima IIPLC-32) immobilized in rice straw BC, with higher activity at pH 3.0. In pinewood nanobiochar, the maximum activity of immobilized Lac (from T. versicolor) was also observed at an acidic pH of 4.0, as reported by Naghdi et al. [44]. However, the residual activity at a near-neutral pH, for both composites, likely reflects partial denaturation under the conditions used (Lac 5.0 mg/mL and pH 6.5). The higher pH is responsible for disrupting hydrogen bonds and disulfide bridges in fungal laccases, destabilizing their copper centers, leading to conformational unfolding and loss of activity [45].

In addition, the results of Figure 3A showed that the activity of SBG-AC/Lac was higher than that of SBG-BC/Lac at the pH of highest activity (pH 3.5), which is related to the higher % of immobilization and activity previously observed using the highly porous SBG-AC as support. Regarding relative activity (%) (Figure 3B), it was observed that Lac_f_ presented the same decreasing trend in activity with increasing pH, but, in general, showed higher stability to changes in pH than with SBG-BC/Lac. Accordingly, Misra et al. [45] found that when Lac (from T. versicolor) was immobilized on another support material (epoxy functionalized polyethersulfone), it had a lower range of pH tolerance compared to Lac_f_, and was deactivated at a pH of 7.4. At the same time, Lac_f_ remained active up to pH 8.0. However, other studies found a different impact of pH on the stability of Lac_f_ and Lac_i_. For example, using Lac (from T. versicolor) and chitosan microspheres as support, Aricov et al. [46] observed that the maximum activity of Lac_f_ and Lac_i_ occurred at pH 3 and 3–5, respectively. In contrast to the results observed in the present work, the authors reported that there was still 10% of the activity of Lac_i_ remaining at pH 7, while the free enzyme was completely inactivated. The overall improved catalytic activity of Laci was related to the support material’s ability to protect the active site from protonation/deprotonation, acting as a buffered microenvironment [46].

In addition, in the present study, although immobilization did not induce a shift in the optimal pH of Lac, Lac_i_ maintained significantly higher activity over time than the free enzyme (cf. Figure 4), confirming the stabilizing effect of immobilization. This behavior is consistent with the literature, which shows that immobilization on carbon-based materials enhances pH tolerance even when shifts in optimal pH are not observed. For example, Zhang et al. [41], using Lac (from Aspergillus) and AC fibers as support material, showed maximum activities of Lac_f_ at pH 4.0, while the optimal activity of Lac_i_ occurred at pH 4.5. Although the authors observed a sharp decrease in the relative activity of Lac_f_ for a pH over 4, the activity of Lac_i_ remained stable over a broad pH range from 3.5 to 6.5. Also, Pandey et al. [25] showed similar results using pine needle BC as support, with Lac_i_ presenting higher stability under pH changes than Lac_f_. Moreover, these authors observed a displacement between the optimum pH of Lac_f_ (pH 3.0) and that of Lac_i_ (pH 4.0).

To evaluate the stability of the free and immobilized Lac under longer contact times, the activity of SBG-BC/Lac (chosen due to its more favorable cost-effectiveness and lower environmental impact compared to SBG-AC/Lac) was investigated periodically, for a period of 24 h of exposure to reaction buffer at the pH of highest activity (3.5). The results of the relative activity % of Lac_f_ in comparison with that of SBG-BC/Lac over time are depicted in Figure 4A.

According to Figure 4A, a decrease in activity (~28% loss of its initial activity) was observed in SBG-BC/Lac immediately after 2 h at pH 3.5 and 40 °C. Then, SBG-BC/Lac mostly maintained its stability for the remaining contact times, with a 36.8% loss of its initial activity after 24 h. In contrast, Lac_f_ lost 46.5 and 96.5% of its activity after 2 and 24 h, respectively, under the same conditions (pH 3.5 and 40 °C). Comparable stabilization effects have been reported in the literature studies for Lac immobilization on agricultural waste-derived carbon materials. For example, Al-Sareji et al. [26], using pomegranate peels AC as support, reported that Lac_i_ presented, in general, slightly higher stability to pH (8% improvement for Lac_i_ compared to Lac_f_), possibly due to the decreased structural pliability and enhanced rigidity, and/or, due to the presence of functional groups on the material support that can scavenge H^+^ from the solution and maintain the enzyme activity. Also, Imam et al. [24], using rice straw BC as support, showed that Lac_i_ maintained its activity (99.9% of its initial activity) after 1 h at pH 3.0. Although in the present work, Lac_i_ showed an initial activity loss during the first hours of exposure, SBG-BC/Lac maintained higher activity than Lac_f_ over extended contact times, as previously referred to, confirming that immobilization onto BC enhances long-term stability under acidic conditions.

2.4.2. Effect of Temperature on Enzyme Activity

The thermal stability of Lac_i_ is important for industrial applications as it enhances its capability to maintain its activity over a range of temperatures, improving its resistance to extreme conditions [47]. A comparison of SBG-BC/Lac activity at two reaction temperatures (20 °C and 40 °C) over time (Figure 4B) revealed distinct trends. At 20 °C, SBG-BC/Lac maintained stable activity during the first 4 h at pH 3.5, followed by a significant decline, losing 63.3% of its initial activity after 6 h. In contrast, at 40 °C, the composite exhibited a rapid decline in activity during the first 2 h but stabilized thereafter, resulting in a comparatively lower activity loss of 24.4% over the same period. According to Naghdi et al. [36], Lac_i_ (from T. versicolor), immobilized onto oxygen-functionalized nanobiochars, showed higher activity at 30 °C, in comparison with lower (20 °C) and higher temperatures (40–70 °C). Also, the previous authors observed that Lac_i_ presented higher stability to temperature than Lac_f_ over the range of 20–70 °C. At temperatures over 50 °C, however, Naghdi et al. [36] found that the inactivation of both Lac_f_ and Lac_i_ occurred due to a higher vibration of the atoms composing the protein, causing drastic changes in its structure and consequently, in its activity. Still, Lac_i_ presented higher stability than Lac_f_, possibly as a result of the decrease in conformational changes of Lac in the support, as also reported by Li et al. [37] for Lac_i_ (using wood BC as support) at pH 4.0 and 60 °C. Indeed, at relatively long contact times (up to 6 h) under these conditions, Lac_i_ maintained 30.3% of its initial activity while Lac_f_ maintained only 10.6% [37]. In accordance, as previously reported by Fortes et al. [42], a rapid decrease in the activity of Lac_f_ (50% of its initial activity after 1.5 h) was observed when compared to Lac_i_ (50% of its initial activity only after 4.1 h) at 60 °C. Other authors also referred to a displacement in the optimal temperature after immobilization, as Al-Sareji et al. [26] observed higher activity of Lac_i_ on AC from pomegranate peels at 30 °C, while for Lac_f_ the optimal temperature occurred at 40 °C. In support materials other than carbon-based ones, Lac_i_ was also reported to present higher stability under temperature changes than Lac_f_. For example, using epoxy-functionalized silica as support, Mohammadi et al. [48] observed higher activity for Lac_i_ than for Lac_f_ at all the studied temperatures (30–45 °C).

2.4.3. Effect of Storage Time and Temperature on Enzyme Activity

The storage stability of immobilized enzymes is another important factor to consider when developing robust biocatalysts, as it affects their usability, economic viability, and efficiency in industrial processes. The activity of Lac_i_ stored under different conditions was also evaluated for SBG-BC/Lac, which showed better cost-effectiveness and environmental friendliness than SBG-AC/Lac, as previously described. This composite was kept at room temperature (in a desiccator) and at 4 °C (in a fridge) for 30 days, and its activity was determined periodically to evaluate its storage stability. The effects of storage time and temperature on enzyme activity are depicted in Figure 5.

The results showed that SBG-BC/Lac lost ~42% of its initial activity during the first 15 days of storage at 4 °C or at room temperature, while maintaining ~28% of its initial activity after 30 days (Figure 5A). To better understand the loss of activity during the first days, the activity of SBG-BC/Lac was measured daily during 5 days of storage at 4 °C or at room temperature. The results (Figure 5B) showed a significant decrease in activity (~37% of the initial activity) after just 2 days of storage at room temperature. In contrast, the activity of SBG-BC/Lac stored at 4 °C remained relatively stable over 5 days. Yet, no significant differences were observed in the activity of SBG-BC/Lac at either temperature (4 °C or room temperature) at any time of storage. Similar trends have been reported for Lac immobilized on carbon-based supports. Naghdi et al. [36] observed a 31% decrease in Lac_i_ activity on oxygen-functionalized nanobiochars during the first 5 days of storage at room temperature. The authors also reported that Lac_i_ showed greater storage stability than Lac_f_: after 30 days, Lac_i_ retained 15% of its initial activity, whereas Lac_f_ showed no remaining activity. As reported previously, the observed increase in storage stability for Lac_i_ can be attributed to the stabilization of the enzyme by the support, conferring structural rigidity and protection against unfolding and denaturation. In prolonged storage conditions (5 weeks), Lonappan et al. [28] showed that Lac_i_ on 3 different types of BC (pinewood, pig manure, and almond shell) maintained 66–80% of its initial activity, due to the improved resistance capacity of enzymes towards the conformational changes when immobilized. On supports other than carbon-based materials, Lac_i_ also presented higher stability under storage conditions than Lac_f_, as reported by Spinelli et al. [49] using Lac (from T. versicolor) and amberlite beads as support material.

2.5. Kinetics

The oxidation of ABTS is catalyzed by Lac in the presence of molecular oxygen. The initial reaction rates of ABTS oxidation were measured at different substrate concentrations (0.05–1.0 mM) for Lac_f_ (final concentration of 1.38 U/L) and SBG-BC/Lac_._ For these measurements, the previously determined optimal reaction conditions of pH 3.5 and a temperature of 40 °C, were chosen. The results for Michaelis–Menten kinetics of ABTS oxidation using Lac_f_ and SBG-BC/Lac are depicted in Figure 6.

For Lac_f_, the KM value was found to be 0.19 (±0.03) mM and the V_max_ was calculated to be 4.5 (±0.2) µM/min (r^2^ = 0.984). For SBG-BC/Lac, the KM and V_max_ values were 0.5 (±0.1) mM and 4.2 (±0.5) µM/min (r^2^ = 0.950), respectively. The KM value is a key parameter in enzyme kinetics, as it reflects the enzyme’s affinity for substrates, with lower KM values indicating a higher affinity [1]. In this work, the higher KM values for SBG-BC/Lac than Lac_f_ suggest a lower affinity of the composite for ABTS, requiring a higher concentration of this substrate to achieve half the maximum reaction rate. This can result from a diffusional limitation of the substrate or from conformational changes in the enzyme induced by immobilization, slightly reducing catalytic velocity. The observed decrease in substrate affinity after immobilization is consistent with previous studies. However, the magnitude of increase in KM is lower when compared to the literature values. As an example, Li et al. [37], showed that Lac_i_ exhibited a higher KM than the free enzyme, with values of 2.68 and 0.22 mM, respectively. Similarly, Zhang et al. [41] showed higher KM for Lac immobilized onto AC fibers compared to Lac_f_. According to Mohammadi et al. [48], the decrease in enzymatic performance is due to structural changes in the enzyme caused by the immobilization process, which reduces the substrate’s access to the active site due to steric hindrance, substrate partitioning, and decreased protein flexibility.

3. Materials and Methods

3.1. Reagents and Chemicals

The 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, $[eqn]$ 98%), laccase (Lac) from Trametes versicolor (EC 1.10.3.2, ≥0.5 U/mg), sodium acetate anhydrous (CH_3_COONa, ≥99%) for molecular biology, potassium carbonate (K_2_CO_3_, 99.9%), and acetic acid (CH_3_COOH, 100%) were purchased from Merck. The hydrochloric acid (HCl, 37.0%) and sodium hydroxide (NaOH, 99.3%) were acquired from Fluka and José Manuel Gomes dos Santos, respectively. All the solutions in this work were prepared in ultrapure water that was obtained using an Elga Purelab Flex 4 purification water system from Elga (Veolia Water Technologies, High Wycombe, United Kingdom).

3.2. Production and Characterization of Carbon-Based Materials

The SBG-AC and SBG-BC materials were produced according to the procedure developed by Sousa et al. [31,32]. As described previously, these waste-derived adsorbents offer economic advantages (lower production costs associated with waste repurposing and microwave pyrolysis to reduce energy consumption), while exhibiting specific surface areas (SBET) (up to 1405 m^2^/g) and high adsorption capacities for the removal of micropollutants from water that are similar to or higher than commercial adsorbents, as previously demonstrated by Sousa et al. [31,32]. Briefly, SBGs were collected in Brewery Faustino Microcervejeira, Lda (Aveiro, Portugal), and transported to the laboratory. The SBGs were then dried in an oven (105 °C) and ground with a blade mill. The dried material underwent an activation step in the case of AC (activation with K_2_CO_3_ in a 1:2 activating agent/SBG ratio). The impregnated SBGs were stirred in an ultrasonic bath for 1 h and then left to dry at room temperature. No activation was performed for BC preparation. The dried impregnated SBGs (for AC) or the ground SBGs particles (for BC) were carbonized under N_2_ atmosphere in a microwave oven (CEM Phoenix™ AirWave, Matthews, North Carolina, United States) and heated at 15 °C/min to 800 °C, with a residence time of 20 min. The resulting materials were washed with ∼ 1.2 M HCl followed by distilled water until the aqueous leachate reached a neutral pH. The materials were separated from the washing leachates by filtration (filter paper, 7–12 µm, from Macherey-Nagel (Düren, Germany), 222009). They were dried overnight at 50 °C, crushed, and sieved to obtain a fine powder (particle size ≤ 180 µm).

The materials produced (SBG-AC and SBG-BC) were characterized regarding N_2_ adsorption isotherms to determine the specific surface area (SBET) and pore distribution. The SBET of the produced materials was determined using a Micromeritics Gemini VII 2380 instrument at 77 K. Before analysis, the samples were degassed at 120 °C. Nitrogen adsorption–desorption measurements were performed using liquid nitrogen at -196 °C. The total pore volume (Vp) was estimated at a relative pressure of 0.99. The Brunauer–Emmett–Teller (BET) equation [50] was applied within a relative pressure range of 0.001–0.1 to calculate the SBET. The microporosity (W0) was evaluated from the low relative pressure region of the nitrogen adsorption isotherm using the Dubinin–Astakhov equation [51]. The average micropore width (L) was obtained from the Stoeckli–Ballerini equation [52]. The average pore diameter (D) was calculated according to Equation (1).

$$[eqn]$$

3.3. Laccase Immobilization

Laccase immobilization was carried out by suspending 100.0 mg of SBG-AC and SBG-BC in 10 mL of 0.1 M sodium acetate/acetic acid buffer at varying pH levels (3.5, 5.0, or 6.5), containing dissolved Lac (from T. versicolor) at varying enzyme concentrations (1.0, 5.0, or 10.0 mg/mL). To test the effect of pH on immobilization, the pH of the immobilization buffer was varied according to the previously referred values (3.5, 5.0, or 6.5) for the lower enzyme concentration (Lac 1.0 mg/mL). To evaluate the effect of enzyme concentration (1.0, 5.0, or 10.0 mg/mL) on immobilization, the pH of the immobilization buffer was fixed at 5.0. The suspensions of material and enzyme were stirred (magnetic stirrer, 150 rpm) at a controlled temperature of 30.0 ± 0.1 °C for 24 h. After the incubation period, the composites were separated from the aqueous phase by filtration (filter paper, 7–12 µm, from Macherey-Nagel, 222009) and washed 3 times with the appropriate buffer (10 mL each time) to remove any unbound enzyme. Each filtered fraction was collected to determine enzymatic activity and assess the effectiveness of the washing procedure. The resulting composites (SBG-AC/Lac and SBG-BC/Lac) were collected from the filter and dried for 48 h at 25 ± 0.1 °C. To evaluate the effect of storage conditions, glass vials containing 2.00 mg of each of the dried composites were stored either at 4 °C, in the fridge, or at room temperature in a desiccator.

The success of immobilization was expressed as immobilization percentage (%), defined as the difference between the initial amount of enzyme (mg/mL) in the incubation solution used for immobilization and the final Lac concentration, determined as the sum of Lac concentrations in all the washing leachates after filtration. A calibration curve of absorbance per min versus Lac concentration (0.03–0.5 mg/mL) was applied to determine the activity (according to Section 3.4. “Enzyme Activity”) and the enzyme concentration in the leachates.

3.4. Enzyme Activity

The activity (A) of the leachates’ free Lac (from Section 3.3. “Laccase Immobilization”), immobilized Lac (Lac_i_) in SBG-AC/Lac and SBG-BC/Lac, and free Lac (Lac_f_, intended to have similar activity than immobilized Lac to ensure a valid comparison) was determined by monitoring the oxidation of ABTS (as chromogenic substrate) in the reaction mixture, following an adaptation of the method developed by Ander and Messner [53] and applied by Fortes et al. [42]. After oxidation by Lac, ABTS is converted into its stable cationic radical, ABTS^•+^. Each determination was performed in triplicate, and the average values were calculated.

To determine the enzymatic activity in the washing leachates (from Section 3.3), the reaction was initiated by adding the Lac-containing sample (at a proper dilution) to 0.05 mM ABTS (final concentration) in 0.1 M sodium acetate/acetic acid buffer, at pH 5.0. The absorbance was measured at 420 nm (Ɛ_420_ = 3.6 × 10^4^ M^−1^ cm^−1^) following the oxidation of ABTS in a microplate reader (SpectraMax 190, Molecular Devices) at 40 °C over time. The catalytic activity was determined by calculating the slope of the initial linear part of the absorbance versus time graph. The enzymatic activity of Lac_f_ was expressed in U/mL, where U corresponds to the amount of Lac required to oxidize 1 µmol of ABTS per minute, under the conditions of the standard assay, according to Equation (2):

$$[eqn]$$

where ∆Abs/min is the value of the slope of the initial linear phase of the kinetic curve, Ɛ_ABTS_ is the molar absorptivity coefficient of ABTS (Ɛ_420_ = 3.6 × 10^4^ M^−1^ cm^−1^), Vreaction is the total volume in the reaction (mL), L is the optical path (cm), Venzyme is the total volume of Lac in reaction (mL), and 10^3^ is the conversion factor of mol/L into µmol/mL.

For Lac_i_, the reaction was initiated by adding 2.00 mg of the composites prepared in Section 3.3 (SBG-AC/Lac and SBG-BC/Lac) to 0.4 mM ABTS (final concentration) in 0.1 M sodium acetate/acetic acid buffer, pH 3.5. Additionally, to ensure a valid comparison between the free and immobilized forms of Lac, the activity of the free enzyme (Lac_f_) was deliberately adjusted. The reaction was initiated by adding the enzyme (at a final concentration of 1.38 U/L) to 0.4 mM ABTS (final concentration) in 0.1 M sodium acetate/acetic acid buffer, pH 3.5. The referred activity of Lac_f_ was determined based on the results of the activity obtained for Lac_i_. In both cases, the mixture was stirred (orbital, 350 rpm) for 8 min in an incubator at 40 °C. After the incubation time, the reaction mixture was filtered through polytetrafluoroethylene (PTFE) hydrophilic syringe filters (0.2 µm, Labfil (Shaoxing, Zhejiang, China), C0000606), and 300 µL of the filtrate was transferred to a 96-well microplate. Similarly to Lac_f_, the absorbance was read at 420 nm in a microplate reader, but using endpoint readings. The enzymatic activity of Lac_f_ was expressed in U/mL as referred to previously, while the activity of Lac_i_ was expressed in U/g dry weight (DW), where U corresponds to the amount of Lac required to oxidize 1 µmol of ABTS per minute, under the conditions of the standard assay, according to Equation (3):

$$[eqn]$$

where ∆Abs/min is the value of the slope of the initial linear phase of the kinetic curve, Ɛ_ABTS_ is the molar absorptivity coefficient of ABTS (Ɛ_420_ = 3.6 × 10^4^ M^−1^ cm^−1^), Vreaction is the total volume in the reaction (L), L is the optical path (cm), mcomposite is the mass of the composite (g), and 10^6^ is the conversion factor of mol/L into µmol/L.

For control purposes, 2.00 mg of the respective composites prepared in Section 3.3, modified to contain inactivated Lac, were used. For inactivation, the composite samples were placed in an oven at 105 °C for 24 h to ensure the enzyme’s denaturation. After this period, the enzymatic activity was determined as described above for Lac_i_. The signal from these controls, which was significantly lower than that of Lac_i_, was subtracted from the corresponding results to assess only the activity of the functional enzyme on the composites.

The results of enzymatic activity were also expressed as relative activity percentage (%). This percentage corresponds, for each evaluated parameter, to the percentage of the activity observed for each condition tested (e.g., reaction pH), both for Lac_i_ and Lac_f_, setting the maximum value of the respective condition at 100%, according to Equation (4):

$$[eqn]$$

where A is the activity of Lac_i_ or Lac_f_ for the condition to be compared and Amax is the maximum value, set at 100% for that condition.

The data obtained for the enzymatic activity from each tested condition were submitted to an ordinary two-way ANOVA, based on Tukey’s multiple comparison, except for the results of the effects of pH on the immobilized activity over time, where an ordinary one-way ANOVA was applied. The software GraphPad Prism 9.5 was used for these analyses. Results with p-values lower than 0.05 were considered significantly different, corresponding to a 95% confidence level.

3.5. Stability of Free and Immobilized Laccase

The stability of Lac_f_ and Lac_i_ was studied in relation to the operation pH, operation temperature, and storage conditions (namely storage time and temperature). Concerning the stability of Lac_f_ and Lac_i_ (SBG-AC/Lac and SBG-BC/Lac) under operation pH, the activity test with ABTS (Section 3.4) was conducted in 0.1 M sodium acetate/acetic acid buffer, with a pH ranging from 3.5 to 6.5 at 40 °C. Afterwards, at a fixed operation pH (3.5) and temperature (40 °C), Lac_f_ and Lac_i_ were maintained at those conditions for 2, 4, 6, and 24 h, and the activity was evaluated at each time point, as described in Section 3.4.

Concerning the stability to temperature, the composites were maintained at 20 °C in 0.1 M sodium acetate/acetic acid buffer (pH 3.5) for 2, 4, and 6 h, and the corresponding activities were compared with those obtained at 40 °C for the same time points. To evaluate the storage stability of the composites, they were stored as previously referred to (Section 3.3), at a temperature of 4 °C in the fridge, or at room temperature in a desiccator. The enzymatic activity was determined immediately after the immobilization procedure, and then after 15 and 30 days of storage at both conditions, according to Section 3.3. Additionally, for SBG-BC/Lac, the activity was further investigated during the first 5 days of storage at both conditions.

3.6. Kinetics

The Michaelis–Menten kinetic parameters (KM and Vmax) of Lac_f_ and Lac_i_ (only SBG-BC/Lac) were investigated by measuring the initial velocities of the reaction with ABTS, with concentrations ranging from 0.05 to 1.0 mM, prepared in 0.1 M sodium acetate/acetic acid buffer, at pH 3.5. The enzymatic activity for the free and immobilized enzymes was determined as described in Section 3.4.

The velocity of the reaction ( $[eqn]$ ) was expressed in μM/min using Equation (5):

$$[eqn]$$

where ΔAbs/min is the value of the slope of the initial linear phase of the kinetic curve, Ɛ_ABTS_ is the molar absorptivity coefficient of oxidized ABTS (Ɛ_420_ = 3.6 × 10^4^ M^−1^ cm^−1^), L is the optical path (cm), and 10^6^ is the conversion factor of mol/L into µmol/L.

The Michaelis–Menten Equation (6) was adjusted to the obtained values using the software GraphPad Prism 9.5 for the nonlinear regression, with posterior determination of KM and Vmax.

$$[eqn]$$

where $[eqn]$ is the velocity of reaction (µM/min), Vmax is the maximum velocity of reaction (µM/min), KM is the Michaelis constant (mM), and [ABTS] is the concentration of substrate oxidized formed during the catalysis (mM).

4. Conclusions

In this work, the optimal conditions for the physical immobilization of Lac (from Trametes versicolor) on two distinctive carbon-based materials, derived from microwave pyrolysis of SBG, namely SBG-AC and SBG-BC, were determined. At their respective best pH of immobilization (pH 5.0 and 6.5), SBG-AC provided higher immobilization of Lac (80 ± 6%) than SBG-BC (58 ± 6%), but that was not reflected, however, in a significant gain in the activity of the corresponding composite. The maximum activities were observed at pH 3.5 and 40 °C, with values of 5.5 ± 0.2 and 4.6 ± 0.5 U/g for SBG-AC/Lac and SBG-BC/Lac, respectively. Besides pH and Lac concentration, other factors, such as the adsorptive properties of the materials, may have accounted for the results observed in the immobilization percentage and activity. Given the similar activity of the two composites, it is worth noting that SBG-BC is a more cost-effective and greener choice due to the absence of chemical activating agents in its synthesis. For this reason, SBG-BC/Lac was further tested, revealing that it presented an improved stability over the free enzyme, maintaining its activity over longer durations (SBG-BC/Lac maintained ~60% of its initial activity after 24 h while Lac_f_ was almost inactivated after that time). In addition, the results showed that SBG-BC/Lac maintained its activity for 5 days of storage at either room temperature or 4 °C. This lab-scale study employed model substrates (ABTS) under controlled conditions, without testing real wastewater conditions or reuse, limiting direct industrial translation. However, this work supports the application of SBG-BC/Lac in industrial applications such as wastewater treatment plants for the elimination of contaminants, as the conventional adsorbents usually present issues related to saturation and regeneration. Combining biochar’s adsorption and Lac’s catalytic activity enables the concentration and degradation of contaminants, reducing operational costs and waste. However, life cycle assessment and pilot-scale validation studies are necessary next steps.