Immobilization of Laccase in β-Cyclodextrin Composite Hydrogel for Efficient Degradation of Dye Pollutants

Abstract

A stable and efficient biocatalyst was prepared by encapsulating Trametes versicolor laccase using an acrylic acid-grafted β-cyclodextrin hydrogel (Lac-CD-PAA). Scanning electron microscopy and nitrogen adsorption-desorption experiments showed that there were regularly distributed channels in the spongy Lac-CD-PAA. In addition, a large number of mesopores and macropores existed in the wall of the hydrogel lamellae. This network structure reduced the diffusion resistance of the hydrogel to the target substrate. The relative activity of the resulting Lac-CD-PAA could be maintained at 35.8% after six cycles of use. Lac-CD-PAA exhibited higher thermal and chemical stability compared to free laccase. The negative charge on the surface of Lac-CD-PAA gives it the ability to pretreat cationic dyes. In six consecutive methylene blue decolorization tests, Lac-CD-PAA decolorized better than free laccase. The results showed that the prepared β-cyclodextrin-based composite hydrogel was a good carrier for laccase.

1. Introduction

With the rapid development of the industry in recent years, the world's ecological environment has been greatly affected [1,2]. Wastewater containing different types of synthetic dyes produced by hundreds of industries, including leather, plastics, textiles, food processing, printing, and cosmetics, were discharged into the natural environment [3]. According to professional statistical data, the annual discharge of dyes in the global industrial sector can reach 30,000−150,000 tons [4,5]. The chemical structure of synthetic organic dyes is usually very complex and difficult to decompose using traditional methods, and if discharged directly into the water body without treatment, their own high chromaticity reduces translucency in the water body, affecting photosynthesis and the respiration of plants and animals in the water body, causing plant and animal death, thereby destroying the ecological system. Synthetic organic dyes are also inducible and carcinogenic, posing serious health hazards to the human body [6,7,8]. Among the types of organic synthetic dyes, ammonia dyes account for the largest proportion, and methylene blue is a type of azo dye, which is widely used in the production of products such as chemicals, dyes, and chemical indicators, etc. Every year, its application in the industrial field generates a large amount of dye wastewater, which is highly difficult to decompose after discharge, highly polluting, and highly hazardous, among other characteristics. The application in the industrial field generates a large amount of dye wastewater every year. Therefore, more effective and safer methods to remove methylene blue dye in water are attracting attention. Immobilized hydrogel is highly absorbent; the hydrogel can quickly absorb a large amount of water and increase its own weight by more than 100 times, which helps to effectively remove water and pollutants in wastewater treatment. Water retention: Hydrogels have excellent water retention properties and can maintain the humidity of an object for a long time, which is especially important for wastewater treatment processes that require a certain level of humidity. Good chemical and physical stability: Hydrogels have a stable molecular chain structure that maintains the water they draw in different environments, with high durability for a wide range of wastewater treatment environments. Environmentally friendly hydrogels are usually made from natural or synthetic polymers, which have a low environmental impact and can be recycled.

Bio-extinguishing methods have attracted attention as an environmentally friendly method of dye degradation [9,10]. Laccases belong to multi-copper oxidases (MCOs) and can oxidize more than 250 substrates, including phenols, aromatic amines, and polyphenols, and catalyze the formation of non-toxic by-products. Several scholars have investigated and studied the degrading effects of laccases on azo dyes [11]. According to the relevant literature, from the degrading effect of laccase on eight dyes, including malachite green violet, methylene blue, and rhodamine B, it was found that although laccase has advantages, such as efficient catalytic activity and good substrate selectivity and specificity [12], it has poor circulating availability of free laccase. However, it was found that free laccase is difficult to put into actual industrial production on a large scale due to its disadvantages, such as easy deactivation and low operational stability [13]. The immobilization of enzymes can effectively solve this problem. Common immobilization methods include embedding, physisorption, covalent bonding, and chemical cross-linking. Among them, the embedding method is a physical method of embedding the guest enzyme in the host substrate, which can more economically and effectively enhance the thermostability and storage stability of the enzyme [14,15,16].

The performance of polymer monomers used in traditional enzyme embedding methods is single and has certain limitations. Therefore, grafting is an attractive way to impart more properties to polymers with various functional groups, where the monomer is attached to the prepolymer monomer chain by covalent bonds [17]. Graft copolymers combine the advantages of the original prepolymer monomer and the newly synthesized polymer, usually with high mechanical properties and good stability [18]. The high electrophilicity of hydrogels can be achieved by selecting prepolymers with electrolyte properties or those containing abundant functional groups such as amines, carboxylic acids, hydroxyl groups, and sulfonic acid groups in the polymer chain [19]. β-cyclodextrins (β-CD) represent a seven-glucose basic unit, a cyclic structure formed by glycosidic linkages, and contains abundant internal chemical bonds such as O-H, C-C, and C-O. It is a unique structure that allows β-CD to form polymers with a variety of organic and inorganic molecules through Van der Waals force and hydrogen bonding action and is an inexpensive, biodegradable, and environmentally friendly polymeric material with high water content and good biocompatibility that can be used as a substrate for enzyme embedding [20,21,22]. Polyacrylic acid hydrogel is a polymeric inexpensive material with special properties and has been widely applied in agriculture, forestry, and medicine; polyacrylic acid hydrogels show the prospect of wide application in many fields with its unique properties [23]. The β-cyclodextrin monomer hydrogel and acrylic monomer hydrogel materials both have remarkable advantages, which are of great significance in the development of new materials that simultaneously possess the performance of grafted molecules and natural polymer polymers and have attracted the attention of researchers.

Graft copolymers are suitable for embedding bioactive substances such as proteins, enzymes, and antibodies in hydrogel substrates, and their immobilized hydrogel structure provides a protective and stable environment that prevents the degradation and inactivation of the bioactive substances and allows for excellent enzyme stability and re-usability [24,25,26]. The encapsulation method is usually associated with encapsulating the enzyme in a porous carrier or polymer gel, and this structure may encounter spatial obstacles in the penetration of the substrate into the mesh or capsule, especially for polymeric substrates, making the encapsulation method potentially unsuitable for enzymatic degradation reactions on polymeric substrates. Although the encapsulation method can retain some enzyme activity, the enzyme activity recovery is generally around 30%, which is relatively low. This may be related to changes in the properties of the enzyme, such as structural changes in the enzyme during the embedding process, the disruption of the active site, and substrate specificity [27]. These are all important parameters that determine the properties of immobilized enzymes. Therefore, a key issue in enzyme immobilization is the design of the hydrogel network structure. Chitosan-grafted polyacrylamide hydrogel (Lac-PAM-CTS) was used as a carrier to produce a stable and highly efficient amphiphilic hydrogel as a carrier for the immobilization of laccase. Such carriers have simultaneous hydrophilic and hydrophobic structures, which play a decisive role in the adsorption performance of certain substrates that can bind water by hydrophilic action and retain solutes dissolved in water by hydrophobic action Lac-PAM-CTS hydrogels, which have a spongy macroporous structure; as a result, the immobilized laccase activity remained above 90%, with a recovery activity of 40.8%. Under the cooperative action of the sponge pore structure and amphiphilic structure of the graft copolymer, good action on contaminant adsorption was achieved. The above results indicate that hydrogels with amphiphilic and spongy structures are the ideal choice for immobilized enzymes [28].

To solve the problem of free enzymes being easily inactivated and not recyclable, β-CD and acrylic acid were selected as the building modules in this study to produce hydrogels of immobilized laccase, taking into account that β-CD and acrylic acid have the advantages mentioned above. First, dissolved laccase was added to the β-CD and acrylic acid mixture and graft copolymerized by radical polymerization, and then N,N'-methylenebisacrylamide (MBA) was added for cross-linking under mild conditions. The prepared large-hole hydrogels with a three-dimensional network structure can diffuse substrate molecules by the active center of immobilized laccase. Taking advantage of the stability and reusability of immobilized laccase bioaccumulators, synthesized composite hydrogel-embedded laccase was used for decolorization experiments on the azo dye methylene blue, and the decolorization process included adsorption of the hydrogel and oxidative degradation of the laccase for the effective removal of methylene blue contaminants from the solution (Scheme 1).

2. Results and Discussion

2.1. Structural Characterization and Analysis of LAC-CD-PAA

2.1.1. FTIR-ATR Analysis

From the FTIR-ATR results, as shown in Figure 1, CD-PAA, Lac-CD-PAA, and CD exhibited the same IR absorption bands near 3435 and 2920 cm⁻¹, corresponding to the stretching vibrations of -OH and -CH-, respectively. In addition, an infrared absorption band near 1160 cm⁻¹ was observed in the spectrogram, which was attributed to the fact that cyclodextrins consist of multiple glucose molecules, leading to the observation of characteristic absorption peaks in the infrared spectra for the sugar ring C-C. In addition, the IR absorption band observed near 937 cm⁻¹ was characteristic of glucopyranose in the cyclodextrin structure, indicating that the modified hydrogel material retained the basic structure of cyclodextrins. In addition, the strong IR absorption peak at 1719 cm⁻¹ indicated the stretching vibration of C=O groups in PAA and MBA [29]. These results indicated that CD, PAA, and MBA were linked together by the free radical polymerization graft copolymerization reaction. In addition, we observed IR spectrograms after immobilization of the enzyme by the composite hydrogel material, which showed IR absorption peaks near 1406 and 1530 cm⁻¹, successfully confirming that laccase was encapsulated in the hydrogel by hydrogen bonding, which enhanced the IR absorption.

2.1.2. X-ray Diffraction Analysis

We know that the diffraction analysis of X-rays to determine the structure of substances is a means often used in experiments, which uses diffraction to carry out quantitative and qualitative analysis so that the results of experiments are more accurate, greatly improving the efficiency of experiments. β-CD, CD-PAA, and Lac-CD-PAA XRD patterns are shown in Figure 2. There were very clear characteristic diffraction peaks at 10–27° in the spectrogram of β-CD. β-Cyclodextrin is a cyclic oligosaccharide whose glucose units are all bonded to form a ring with α-1,4-glycosidic bonds. Since the glycosidic bonds connecting the glucose units cannot rotate freely, β-cyclodextrin is not a cylindrical molecule but a slightly tapered ring. A broad characteristic diffraction peak appeared at 22.5° in the spectrogram of CD-PAA, Lac-CD-PAA, indicating that both the modified CD hydrogel material and the hydrogel material after immobilized laccase belonged to an amorphous phase-disordered structure. These results suggest that β-CD was modified to increase the amorphous nature of the polymer, transforming the polymer from a crystalline to an amorphous state [30].

2.1.3. Thermogravimetric Analysis

The thermal stability properties and applicable temperatures of β-CD hydrogels modified by PAA were analyzed by the thermal weight loss assay (TGA), and the results are shown in Figure 3. We observed the fact that the thermal decomposition phase of β-CD is mainly divided into three stages. The first stage of decomposition occurred in the temperature range of 50–100 °C with a weight loss rate of approximately 12.2%, which is attributed to the volatilization of physically bound water within the β-CD cavity. The second stage underwent a sharp decomposition in the range of 300–350 °C with a weight loss rate of approximately 78.2%, indicating that the main skeletal structure of β-CD decomposed and some residual material was retained. The third stage occurred slowly in the 350–600 °C range, which was attributed to the continued decomposition of residual substances in β-CD [31]. The overall primary decomposition phase of CD-PAA hydrogels was significantly longer, the decomposition rate was significantly lower than that of β-CD, and the decomposition temperatures were concentrated in the temperature range of 300–450 °C, which was mainly attributed to the thermal decomposition or volatilization of organic small molecules and residues in the PAA moiety. The analysis showed that the CD-PAA hydrogels had better thermal stability and lower weight loss compared to β-CD.

2.1.4. Surface Morphology Analysis

The SEM analysis of the surface morphology of CD-PAA and Lac-CD-PAA composite hydrogels is shown in Figure 4. Fresh CD-PAA and Lac-CD-PAA composite hydrogels are clear, homogeneous, and transparent, and by soaking and rinsing them in deionized for three times, unreacted components, including oligomers and even laccase molecules, can be washed away from the open channels. After freeze-drying using a lyophilizer, SEM tests were performed, and as can be observed in Figure 4a,a’, the surface of the sample was rough, irregular, and uneven, and the channels in the three-dimensional network structure, where only water existed originally, were empty, with a clear porous structure, and the presence of micropores of varying sizes. The internal structure of the composite hydrogel showed a complex three-dimensional porous reticulated lamellar structure, which produced micropores of varying sizes in the lamellar walls of the channels. The large number of interpenetrating micropores provided a large number of active adsorption sites, which facilitated the adsorption of methylene blue. From Figure 4b,b’, it can be observed that the composite hydrogel has a large amount of laccase wrapped around the lamellar wall of the channel and still has a large number of microporous structures while not affecting the physical adsorption of methylene blue. Therefore, using CD-PAA hydrogel to immobilize laccase, a biocatalyst that can physically adsorb methylene blue and oxidatively degrade methylene blue, at the same time, was obtained.

2.2. Swelling Kinetic Characteristics

Swelling dynamics is one of the key parameters in hydrogel, which directly affects its use and performance. Therefore, analyzing the swelling kinetic properties of hydrogels is of great significance to deeply understand their performance and promote the development of their applications. Hydrogel polymers with both hydrophilic and hydrophobic amphiphilic networks were prepared by introducing hydrophilic group-COOH (from PAA) and hydrophobic group -CONH₂ (from MBA) into β-CD. Hydrogels containing both hydrophilic and hydrophobic structures swell less in water than single hydrophilic hydrogels, but they can swell in organic solvents [32]. As shown in Figure 5a, both in water and toluene, there was first rapid swelling and then a slow plateau. In hydrogels, the interaction of hydrophilicity and hydrophobicity determined the manifestation of amphiphilicity. When the hydrogel was in a dry state, the hydrophobic groups were close to each other, causing the hydrogel to shrink in size. And when the hydrogel was in contact with water, the hydrophilic groups interacted with water molecules, causing the hydrogel to absorb water and swell. Subsequently, the swelling of the hydrophobic units weakened or hardly occurred, and the swelling of the hydrophilic units weakened, eventually entering a relatively slow phase to form a stationary phase [33]. This swelling mechanism also applies to the swelling in toluene, in contrast to the hydrophilic unit, i.e., the toluene molecule first caused the hydrophobic unit to swell, then the hydrophilic unit, and then the swelling gradually weakened to equilibrate into the stationary phase. Droplet contact angle tests further confirmed the amphiphilic nature of Lac-CD-PAA (Figure 5b). Compared with homopolymerized hydrogels, Lac-CD-PAA, with an amphiphilic nature, demonstrated better mechanical properties due to the presence of hydrophobic structural domains, which reduced its water content and made its structure more stable.

2.3. Properties and Stability of Free and Immobilized Laccase

Environmental stability is one of the most important characteristics of biocatalysts for practical applications. We determined the relative residual activities of free and immobilized laccase at different pHs (3,4,5,6,7,8) and 65 °C to illustrate their stability to changes in environmental conditions. As shown in Figure 6a, the catalytic activity of free laccase was almost undetectable when the solution was under neutral or alkaline conditions, while residual activity remained in Lac-CD-PAA. As shown in Figure 6b, after 48 h of incubation, the loss of activity of Lac-CD-PAA was smaller than that of free laccase, which retained only 4.3%. Therefore, the better stability of Lac-CD-PAA compared with free laccase could be attributed to the protection of laccase by the support matrix and the buffering effect against the external environment.

While the increased resistance of immobilization to changes in environmental conditions is advantageous for the industrial application of laccase in the reaction of free versus immobilized laccase for ABTS, K_m is the most useful kinetic parameter in the Michaelis–Menten model for assessing the enzyme–substrate binding affinity [34]. The Mie kinetic parameters of free laccase and Lac-CD-PAA are shown in Figure 7 and

Table 1. Parameters obtained from fitting the Lineweaver–Burk plot.

Samples	R2	Vmax (μmol/(L · min))	Km (μmol/L)
Free laccase	0.9785	352.11	219.90
Lac-CD-PAA	0.9813	432.90	140.92

. We can observe that the reaction rate constant K_m of the immobilized laccase is smaller than that of free laccase, which may be attributed to electrostatic attraction, whereby the substrate concentration in the microenvironment of the immobilized enzyme is higher than in the overall solution, resulting in a decrease in the apparent K_m value of the immobilized enzyme. In this case, the enzyme binds more readily to the substrate, exhibiting an increased affinity for the substrate. In addition, the maximum reaction rate V_max of the immobilized laccase is also greater than that of free laccase, which is a phenomenon that occurs because of the increased affinity of the substrate for the immobilized enzyme and the increased concentration of the substrate around the enzyme, thus simultaneously increasing the rate of the reaction catalyzed by the enzyme.

2.4. Decolorization Experiment of Methylene Blue by Lac-CD-PAA

The effect of the mass concentration of methylene blue on the removal effect was investigated by placing 50 mg dry-weight Lac-CD-PAA after solubilization in different mass concentrations of the methylene blue solution (5–20 mg/L). As shown in Figure 8a, when the initial mass concentration of methylene blue was 5 mg/L, the removal effect of Lac-CD-PAA could reach 95.2% at 1 h. With the gradual increase in the mass concentration of the methylene blue solution, the time for the removal effect to reach 95.2% also increased gradually. When the mass concentration of methylene blue was 15 mg/L, it took 8 h for Lac-CD-PAA to completely remove the methylene blue, and when the mass concentration of methylene blue was 20 mg/L, it took 12 h for Lac-CD-PAA to completely remove the methylene blue. Considering the economic benefits of the actual application and the change in the activity of laccase, the mass concentration of methylene blue was set at 15 mg/L for the present experiment.

The solubilized 50 mg dry weight free laccase, Lac-CD-PAA, and LosLac-CD-PAA were placed in the methylene blue solution, and the mass concentration of methylene blue in the solution was measured at regular intervals to observe the removal effect of the three materials on methylene blue. From Figure 8b, it can be observed that the removal effect of the three materials on methylene blue was Lac-CD-PAA>LosLac-CD-PAA>free laccase, and the decolorization effect of Lac-CD-PAA on methylene blue was slightly higher than that of LosLac-CD-PAA, which indicated that not only adsorption but also enzymatic degradation existed in the Lac-CD-PAA system. In the LosLac-CD-PAA system, the decolorization of methylene blue was exclusively from the electrostatic adsorption of the cationic dye methylene blue on the anionic hydrogel carrier matrix. In other words, the electrostatic attraction from the charged hydrogel may have a pre-enrichment effect on the target contaminant. This pre-enrichment effect may increase the substrate concentration of the immobilized laccase, thereby enhancing the degradation of the target pollutant.

2.5. Decolorization Experiment of Methylene Blue by Lac-CD-PAA

The reusability of biocatalysts is an extremely important evaluation indicator of whether they can be popularized in practical applications. As shown in Figure 9, to evaluate the performance of immobilized laccase, free laccase and Lac-CD-PAA were placed in methylene blue solution and incubated repeatedly six times to determine their removal rates from methylene blue. After six repetitions, the removal of methylene blue by Lac-CD-PAA was significantly better than that of free laccase, which indicated that the recovery rate of Lac-CD-PAA was greatly improved, although the adsorption sites on the material were gradually saturated with the continuous adsorption of methylene blue, and laccase could oxidize and decompose methylene blue to restore the adsorption sites, allowing methylene blue to be removed continuously and effectively [35].

3. Materials and Methods

3.1. Materials

β-cyclodextrin (β-CD, 98%) and laccase from Trametes versicolor (0.5 U/mg) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Acrylic acid (AA), ammonium persulfate (APS), and N,N-methylenebis (acrylamide) (MBA) were all of analytical grade and were obtained from J&K Scientific Co., Ltd, (Beijing, China). Sodium hydroxide (NaOH, A.R.) and hydrochloric acid (HCl, A.R.) were obtained from National Pharmaceutical Group Chemical Reagent Co., Ltd. (Beijing, China). All reagents used were as received, and all aqueous solutions were prepared using deionized water prepared in a laboratory ultrapure water machine.

3.2. Preparation of Hydrogel-Entrapped Laccase (Lac-CD-PAA)

The optimal preparation process of hydrogel-entrapped laccase was as follows: 1 g of β-cyclodextrin (β-CD) powder was dissolved in 20 mL of water, heated at 50 °C, stirred for 10 min until all of it was dissolved, and 50 g/L of β-CD solution was prepared. A 70% AA solution was prepared by taking 50 mL of pure acrylic acid (AA) monomer and mixing it with 30 mL of NaOH solution (20 wt%). In total, 7.2 g of 70% AA solution was added to a single neck flask along with 20 mL of β-CD solution and stirred to mix well. Then, 0.1 g of MBA crosslinker was added to the flask and mixed well, and stirring was continued until all the mixture was dissolved and cooled to room temperature. To the flask, we continued to add 0.1 g/mL of the initiator ammonium persulfate (APS) at 5 mL and mixed well at 150 rpm and room temperature. The above mixed homogeneous solution was poured into a mold and placed in an oven at 40 °C for 2 h until it completely became a hydrogel. The prepared hydrogels were washed three times with deionized water to remove unreacted raw material components and small molecular compounds. The washed hydrogels were dried with a freeze dryer (BUCHI Labortechnik AG, Model: BUCHI-L200) for 48 h. The prepared hydrogel-entrapped laccase composite (denoted as Lac-CD-PAA) was cut into blocks (2 mm × 2 mm) and stored in a −20 °C refrigerator for use in the following experiments. A portion of Lac-CD-PAA was soaked in methanol overnight to inactivate laccase, and the inactivated Lac-CD-PAA was recorded as LoseLac-CD-PAA.

3.3. Characterization

The morphology of Lac-CD-PAA was studied by scanning electron microscopy (SEM) (ZEISS Company, Jena, Thuringia, Germany). The samples were imaged in ultra-high vacuum mode at an accelerating voltage of 5 kV using a SEM. The samples were placed on monocrystalline silicon wafers and, after freezing in liquid nitrogen, were rapidly transferred to a freeze dryer for 12 h. The dried samples were carefully placed on a conductive adhesive and then coated with gold vapor to make them conductive, followed by an SEM examination. Fourier transform infrared spectra ATR (FTIR-ATR) analysis was conducted using PerkinElmer FTIR Spectrometer Spectrum Two (PerkinElmer Company, Waltham, MA, USA). The FTIR-ATR spectrum was acquired in the scanning range of 4000−400 cm⁻¹ with a resolution of 4 cm⁻¹. The thermogravimetric analysis measurements were performed on a HITACHI STA200 simultaneous thermal analyzer (Hitachi Company, Tokyo, Japan), with a heating rate of 10 °C/min from room temperature to 500 °C in a (N₂, Air, Ar) atmosphere. The morphological characteristics of Lac-CD-PAA hydrogels were observed by X-ray diffraction (XRD). The droplet contact angle of Lac-PAM-CTS was measured using a SZ-CAMC33 contact angle meter (Shanghai Xuanzhun Company, Shanghai, China).

3.4. Study of Swelling Properties of Hydrogels

To study the swelling of the prepared Lac-CD-PAA hydrogels, dried block samples (2 mm × 2 mm) were weighed (noted as m₀) and immersed in deionized water and toluene, respectively, at room temperature. The swollen samples were weighed (noted as m_t) after excess water or toluene was wiped off the surface of the samples at certain time intervals with filter paper. The swelling rate test was averaged over three times for each set of experiments. The swelling rate (S) was calculated as shown in Equation (1).

$$\mathrm{S}={\displaystyle \frac{({\mathrm{m}}_{\mathrm{t}}-{\mathrm{m}}_0)}{{\mathrm{m}}_0}}\times 100\%$$

(1)

Here, m_t (g) represents the mass of the hydrogel after swelling at time t, m₀ (g) represents the mass of the dried hydrogel before the experiment, and S (%) represents the swelling ratio of the hydrogel.

3.5. Determination of Laccase Activity

Free laccase and immobilized laccase activities were determined separately using 1 mM of ABTS as the substrate. The reaction was initiated by adding 1 mL (5 mg/mL) of laccase solution to a cuvette, followed by 1 mL of acid-sodium acetate (pH 5.0, 200 mM) buffer solution, and 1 mL (1 mmol/L) of ABTS solution to the cuvette at room temperature, and the change in absorbance value at 420 nm for 1 min of the reaction was measured with a UV-visible spectrophotometer. The laccase activity unit (U) was defined as the amount of the enzyme oxidizing 1 μmol of ABTS per minute under the experimental conditions [36], and the formula for calculating the enzyme activity of laccase is shown in Equation (2). The enzymatic activity of immobilized laccase was determined in the same way as that of the free laccase, but Lac-CD-PAA (20 mg) was used instead of free laccase solution.

$$\mathrm{U}={\displaystyle \frac{({\mathrm{A}}_{\mathrm{t}}-{\mathrm{A}}_0)\times {\mathrm{V}}_1\times {10}^3}{\mathrm{t}\times {\mathrm{V}}_2\times \mathsf{\epsilon }}}$$

(2)

Here, A_t represents the absorbance at time t, A₀ represents the initial absorbance, V₁ (mL) represents the total volume of the solution, V₂ represents the volume of laccase solution added, and ε represents the molar extinction coefficient of ABTS⁺ in 360 mmol⁻¹ · cm⁻¹.

3.6. Michaelis–Menten Kinetic Parameters

Using ABTS as the substrate, with initial concentrations ranging from 0.4 to 10.0 mM, the Michaelis-Menten kinetic parameters K_m and V_max of free and immobilized laccase were measured, and from the Lineweaver-Burk double reciprocal model of the Michaelis–Menten equation K_m, Equation (3) was calculated as follows:

$${\displaystyle \frac{1}{\mathrm{V}}}={\displaystyle \frac{{\mathrm{K}}_{\mathrm{m}}}{{\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}}\times {\displaystyle \frac{1}{\mathrm{S}}}+{\displaystyle \frac{1}{{\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}}$$

(3)

Here, V_max (μmol/(L·min)) represents the maximum reaction speed, S (μmol/L) represents the substrate concentration, and K_m (μmol/L) represents the Michaelis constant. Let us draw a straight line from ${\displaystyle \frac{1}{\mathrm{v}}}$to${\displaystyle \frac{1}{\left[\mathrm{S}\right]}}$, where the slope is ${\displaystyle \frac{{\mathrm{K}}_{\mathrm{m}}}{{\mathrm{v}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}}$, the vertical intercept is ${\displaystyle \frac{1}{{\mathrm{v}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}}$, and we know the value of V_max and K_m.

3.7. Stability Test of the Lac-CD-PAA

The stability tests included three sets of tests at different solution pHs and different times. pH stability, the effect of solution pH on the relative enzyme activities of immobilized and free laccase was as follows: Lac-CD-PAA and free laccase (protein content: 1 mg of protein per mL) were incubated in phosphate buffer (50 mM) at different pHs of 3, 4, 5, 6, 7, 8, and 25 °C for 48 h. The changes in the relative activities of immobilized and free laccase were determined by incubating them at different pHs. The stability of immobilized and free laccase at different pH levels was evaluated by incubation at 25 °C for 48 h to determine the changes in the relative activity of laccase at different pHs. For thermal stability at the same temperature, the storage stability of immobilized and free laccase was evaluated by incubating them in phosphate buffer (50 mM, pH 5.0) at 65 °C for 10 h, and the changes in the relative activity of laccase at different times were determined. Each group of experiments was performed three times to take the average value.

3.8. Methylene Blue Decolorization Experiment

Prior to decolorization experiments, Lac-CD-PAA and LosLac-CD-PAA were soaked in deionized water for 4 h to reach a swelling equilibrium. Then, 50 mg of dry re-swollen Lac-CD-PAA was added into 5, 10, 15, and 20 mg/L of methylene blue (1 mL, pH = 3) solution, respectively, and then the solid–liquid mixture reactants were incubated at 25 °C, 100 r/min under continuous light protection, and the samples were taken at regular intervals, and then the absorbance of methylene blue in the solution at the maximum absorption wavelength of 662 nm was measured using a UV spectrophotometer and methylene blue The mass concentration of methylene blue was calculated according to the absorbance.

3.9. Experiments on the Recycling of Lac-CD-PAA

Prior to decolorization experiments, Lac-CD-PAA and LosLac-CD-PAA were soaked in deionized water for 4 h to reach a swelling equilibrium. Then, 50 mg of dry re-swollen Lac-CD-PAA was added into 15 mg/L of the methylene blue (1 mL, pH = 3) solution, and the solid–liquid mixed reactants were continuously incubated at 25 °C, 100 r/min, under light protection for 8 h. After that, Lac-CD-PAA was fished out with a strainer and placed in deionized water (pH = 3, 25 °C) for 8 h. The reactants were reintroduced into methylene blue solution to start a new round of decolorization. The methylene blue solution started a new round of reactions, which was carried out for 6 consecutive rounds. Free laccase and LosLac-CD-PAA were used for control experiments in the same way.

4. Conclusions

In this experiment, a Lac-CD-PAA amphiphilic biocatalyst was prepared for the degradation of the organic cationic dye methylene blue using the free radical polymerization crosslinking and grafting method, which is an environmentally friendly crosslinked polymer CD-PAA composite hydrogel carrier material with good mechanical properties and a high recovery rate. Due to the large number of microporous structures and open channels, it has a good pre-enrichment effect on cationic pollutants and good reusability in the continuous decolorization of dye pollutants. The adsorption of the dye by the hydrogel carrier material, combined with the oxidative degradation of methylene blue dye by laccase, means that the decolorization efficiency of Lac-CD-PAA is more than twice as high as that of free laccase. And the relative viability was still maintained at 38.5% after seven rounds of recycling. Overall, the method of making composite hydrogel materials results in a promising immobilization carrier for enzymes, and Lac-CD-PAA, made by the embedding method, is an efficient biocatalyst with high potential for application in wastewater treatment.