A Novel Cysteine-Functionalized M x O y Material as Support for Laccase Immobilization and a Potential Application in Decolorization of Alizarin Red S

: Immobilization process improves the enzyme properties, like stability, activity, selectivity or speciﬁcity. In the study, a novel cysteine-functionalized M x O y (ZrO 2 , SiO 2 ) material was used as a support for the immobilization of laccase from Trametes versicolor . The proposed matrix was prepared using a simple sol-gel method. The cysteine was introduced during the synthesis of a sample. Additionally, the obtained supports were modiﬁed with glutaraldehyde. The basic properties of the prepared cysteine functionalized ZrO 2 and SiO 2 were determined using spectroscopic, thermal, porous, electrostatic and elemental analysis. Furthermore, the obtained biocatalytic systems were used as catalysts in the oxidation of sulfonic acid. Catalytic and kinetic parameters were determined based on the proposed model reaction. Next, laccase immobilized on ZrO 2 - and SiO 2 -based materials were, for the ﬁrst time, utilized in the decolorization of Alizarin Red S. In that process, the inﬂuence of duration, pH and temperature on the e ﬃ ciency of decolorization was evaluated. The results show that the proposed biocatalytic systems o ﬀ er good speciﬁc activity (ca. 19 U / mg) and activity retention (ca. 77%). Importantly, they can be successfully used in the decolorization of Alizarin Red S with high e ﬃ ciency (above 95%).


Introduction
The enzyme immobilization is a powerful tool in biocatalyst design, improving protein properties. A proper immobilization can increase stability and activity of enzyme under conditions far from the physiological ones, enzyme selectivity and specificity (using substrates far from the physiological ones), enzyme purity and sensitivity to inhibition, as well as resistance to chemicals. The support properties, the active group presence in the support and enzyme should be considered in immobilization protocol [1].
Cysteine ((R)-2-amino-3-mercaptopropionic acid, Cys) is a branched amino acid which contains three functional groups: thiol, amino and carboxyl. The thiol groups can be used to create supports with disulfide bonds. The thiol groups can form stable disulfide bonds; they may bind with metals by coordinate bonding or remain in reduced form [2,3]. Besides, thiol groups play an important role in the synthesis and functionality of metal nanoparticles, because of their high affinity to the particle surface [4]. Additionally, cysteine prevents the aggregation of nanoparticles and enables the attachment of enzymes on the particles' surface. The amino and carboxyl groups present in Cys are suitable for the immobilization of enzymes. Furthermore, because of the presence of thiol groups, cysteine is used in the pharmaceutical industry (for drug delivery) and the food industry (as a food additive) [5,6]. Cysteine as a natural antioxidant in several biological processes, including protein derivatives could cause cytotoxicity, genotoxicity and DNA strand breakage [34]. Many methods are used for the degradation and decolorization of dyes. These methods can be divided into three categories: physical methods (nano-filtration, reverse osmosis, electrodialysis) [35], sorption techniques (photochemical, electrochemical destruction) [36] and biological methods (enzymatic degradation) [37]. Biological methods have low running costs, produce stable and harmless final products, and also require fewer chemicals and less energy than physical and chemical methods. Furthermore, enzymatic degradation complies with the principles of green technology [38]. Immobilized enzymes, especially oxidoreductases (laccases and peroxidases), are used to improve decolorization methods. Many dyes-for example, Acid Blue, Reactive Blue, Remazol Brilliant R, Direct Red etc.-have been decolorized using immobilized laccase. For this purpose, MOFs, bacterial nanocellulose, electrospun fibers and graphene oxide have been utilized as supports for the immobilization of laccase [39][40][41][42]. Besides the laccase, the different peroxidases were also successfully used to decolorize organic dye-based wastewaters [43][44][45].
In this study, we propose a novel cysteine-functionalized M x O y material as a support for enzyme immobilization. The materials used are SiO 2 and ZrO 2 , prepared by the sol-gel method. Cysteine was applied in situ, that is, during the sol-gel synthesis. Additionally, the obtained material was activated with glutaraldehyde to improve the attachment of laccase to the material surface. Next, laccase from Trametes versicolor (light, brown powder with activity above 0.5 U/mg) was immobilized on the cysteine-functionalized M x O y by a simple adsorption method. The research included evaluation of physicochemical properties (Fourier-transform infrared spectroscopy, thermogravimetric, porous structure, zeta potential and elemental analysis) and catalytic properties (relative activity, kinetic parameters, influence of pH, temperature, storage and reuse on enzymatic activity). The obtained biocatalytic system was also tested in the decolorization of an organic dye (Alizarin Red S).

Synthesis of l-Cysteine-Functionalized M x O y
The metal oxides (SiO 2 and ZrO 2 ) were synthesized by a sol-gel method. In the first stage, an appropriate alcohol (ethanol for SiO 2 and isopropanol for ZrO 2 ) was introduced into a reactor. Next, the organic precursor (TEOS for SiO 2 and ZIP for ZrO 2 ) and the promotor of hydrolysis (NH 3aq. ) were dosed. Then l-cysteine (10% wt./wt., in 1 M of HCl) was added. The components were mixed for 1 h (at ambient temperature) and left to age for 48 h. The synthesized materials were dried at 105 • C for 12 h. Finally, the obtained powder was washed several times using distilled water and alcohol, and the prepared materials were again dried (12 h, 105 • C). As a result, the systems SiO 2 _Cys and ZrO 2 _Cys were obtained. The precise information regarding sol-gel synthesis is presented in Table 1. In the next step, SiO 2 _Cys and ZrO 2 _Cys were activated with glutaraldehyde (5% in pH = 7 buffer) for 24 h. This led to the systems SiO 2 _Cys_GA and ZrO 2 _Cys_GA.
The four kind of supports (SiO 2 _Cys; SiO 2 _Cys_GA, ZrO 2 _Cys and ZrO 2 _Cys_GA) were used in the next step of research.

Immobilization of Laccase from Trametes Versicolor and Bradford Analysis
Immobilization of laccase from Trametes versicolor was led by adsorption and covalent method. The support (0.5 g of SiO 2 _Cys; SiO 2 _Cys_GA; ZrO 2 _Cys and ZrO 2 _Cys_GA) was added to the laccase solution (25 mL of solution with the concentration of 5 mg/mL in 0.1 M buffer at pH = 4). The immobilization process took place for 3 h at 25 • C in an incubator (IKA-Werke, Staufen, Germany). Bradford analysis was used to calculate the quantity of immobilized enzyme [46]. The quantity of immobilized enzyme (mg/g support ) is determined from the difference between the initial amount of enzyme and the final laccase concentration in the mixture after immobilization. The calculation was made relative to the mass of the support. The quantity of immobilized laccase (P) and immobilization yield (IY) were calculated using Equations (1) and (2): where C 0 and C 1 denote the concentration of the enzyme (mg/mL) in solution before and after immobilization, respectively, V is the volume of solution (mL), and m is the mass of support (g). The following four biocatalytic systems were prepared: SiO 2 _Cys_Lac; SiO 2 _Cys_GA_Lac; ZrO 2 _Cys_Lac and ZrO 2 _Cys_GA_Lac.

Physicochemical Characterization
Spectroscopic, thermogravimetric, porous structure, zeta potential and elemental analysis were applied to characterize the samples obtained during the study.
Contents of carbon, nitrogen, hydrogen and sulfur were evaluated to confirm the effectiveness of modification with cysteine. For this purpose, the Vario EL Cube apparatus (Elementar Analysensysteme, Langenselbold, Germany) was used. The analyzed sample (ca. 20 mg) was combusted in an oxygen atmosphere. After passing through a reduction tube, the resulting gases were separated in an adsorption column, and then recorded using a detector. The results are given as averages of three measurements, each accurate to 0.0001%.
Other analyses were used to characterize samples obtained before and after immobilization. Spectroscopic analysis was performed based on FTIR spectra obtained using a Vertex 70 spectrometer (Bruker, Billerica, MA, USA). The analyzed sample had the form of a tablet, made by pressing a mixture of anhydrous KBr (ca. 0.25 g) and 0.001 g of the analyzed material in a special steel ring under a pressure of 10 MPa.
Basic porous structure parameters of prepared samples were determined using an ASAP 2020 instrument (Micromeritics Instrument Co., Norcross, GH, USA). Before the analysis, all samples were degassed (support at 120 • C and biocatalytic system at 70 • C) for 4 h prior to measurement. Next, based on low-temperature N 2 sorption the analysis was carried out. Using the BET (Brunauer-Emmett-Teller) and BJH (Barrett-Joyner-Halenda) methods, the surface area (A BET ), total pore volume (V p ) and mean pore diameter (S p ) were assessed. Due to the high accuracy of the instrument used, surface area was determined to an accuracy of 0.1 m 2 /g, pore volume to 0.01 cm 3 /g, and pore size to 0.01 nm.
Additionally, the zeta potential and isoelectric point (IEP) were evaluated using the LDV (Laser Doppler Velocimetry) technique, and calculated based on the Henry equation. These parameters were determined using a Zeta Nano ZS equipped with an MPT-2 automatic titration system (Malvern Instruments Ltd., Malvern, Worcester, UK). For the measurement, 0.01 g of the sample was dispersed in 25 mL of sodium chloride solution. Titration was performed with 0.2 M solutions of HCl and NaOH. The standard deviation of the zeta potential measurement was 61.5 mV. The apparatus measures a single zeta potential 30 times at defined pH, and the average value is used as the final result. The standard deviation of the pH value measurement was 0.1.

Catalytic Properties
The influence of pH, temperature, storage stability and reusability on the catalytic activity of a biocatalytic system is the most important information describing an immobilized enzyme.
The influence of temperature was tested in the range 30-70 • C, and the influence of pH in the range 3-7. Storage stability was evaluated after 30 days (the immobilized enzyme was stored in a pH = 4 buffer at 4 • C). Reusability is the most important parameter for an immobilized enzyme; thus, the relative activity was investigated after 10 cycles. For clearer presentation of the data, in these experiments, the highest activity of free and immobilized laccase was defined as 100% activity. All of the above parameters were determined based on the reaction of oxidation of ABTS. In this case, 10 mg of free or immobilized laccase was added to 20 mL of 0.1 mM ABTS. The reaction was carried out for 20 min at 40 • C. Next, the mixture was centrifuged, and the absorbance was measured at λ = 420 nm (V-750 spectrophotometer, Jasco, Oklahoma City, OK, USA). The required parameters to define the immobilization process were determined [47]. The apparent activity of laccase was defined as the quantity of enzyme which oxidized 1 µM of ABTS per minute per 1 g of support. The activity retention and specific activity of laccase immobilized on the support were calculated according to Equations (3) and (4): where A R -retention activity (%); A S -specific activity (U/mg enzyme ); A S1 -specific activity of immobilized Lac (U/mg); A S0 -specific activity of free Lac (U/mg); A Ap -apparent activity (U/g support ); P-amount of immobilized Lac (mg/g). Additionally, the kinetic parameters (K M , the Michaelis-Menten constant; and V max , the maximum reaction rate) were evaluated based on the above-mentioned ABTS oxidation reaction and calculated using Hanes-Woolf plots. In this process, various concentrations of the ABTS solution (0.005-1.5 M) were used.
All measurements were made in triplicate. Results are presented as mean ± 3.0 SD.

Decolorization of Alizarin Red S
A process of decolorization of Alizarin Red S dye was carried out using the four prepared biocatalytic systems. For this purpose, 100 mg of each of the biocatalytic systems was placed in 10 mL of Alizarin Red S dye solution (50 mg/mL; pH = 7). The process was performed at 30 • C for 24 h. The influence of time (0.5, 1, 3, 6, 9, 12, 24 h), temperature (25-70 • C) and the pH of the environment (2-9) on the effect of decolorization of the dye was determined. During these tests, differences in pH in absence of enzyme did not influence dye decolorization. Each experiment was carried out in triplicate, and the results are presented as average values. After each of the above-mentioned experiments the absorbance of the resulting solution was measured (α = 464 nm; V-750 spectrophotometer, Jasco, Oklahoma City, OK, USA). The efficiency of decolorization of the dye was calculated based on the value of absorbance and using Equation (5): where ED is the efficiency of decolorization of ARS; C B and C A are the concentrations of ARS dye before and after the decolorization process, respectively.

Thermogravimetric and Elemental
Analysis of Pure Supports (ZrO 2 _Cys, ZrO 2 _Cys_GA, SiO 2 _Cys and SiO 2 _Cys_GA) The materials used as supports for the immobilization of enzymes should have high thermal stability. Thermogravimetric analysis is one of the methods of evaluating the thermal stability of materials. The TG/DTG curves of pure cysteine show that this material has low thermal stability (Figure 1a,b). Two mass losses are observed: the first at ca. 200 • C related to physically adsorbed water, and the second at ca. 400 • C corresponding to the release of crystallization water. Zirconium and silica oxides offer good thermal stability [48]. As shown in Figure 1, all of the materials prepared in this study have high thermal stability. A mass loss of 20% was observed over the whole analyzed temperature range (30-1000 • C) for ZrO 2 _Cys (Figure 1a). An additional mass loss at 900 • C was observed for the ZrO 2 _Cys_GA system, probably associated with the thermal decomposition of glutaraldehyde. For SiO 2 _Cys and SiO 2 _Cys_GA, the mass loss was smaller than in the case of the zirconium materials. In this case the total mass loss was 10% over the analyzed range of temperatures (30-1000 • C). To summarize these results, it was found that the proposed cysteine-functionalized ZrO 2 or SiO 2 materials are suitable for use as supports in the immobilization of enzymes.

Thermogravimetric and Elemental Analysis of Pure Supports (ZrO2_Cys, ZrO2_Cys_GA, SiO2_Cys and
SiO2_Cys_GA) The materials used as supports for the immobilization of enzymes should have high thermal stability. Thermogravimetric analysis is one of the methods of evaluating the thermal stability of materials. The TG/DTG curves of pure cysteine show that this material has low thermal stability (Figure 1a,b). Two mass losses are observed: the first at ca. 200 °C related to physically adsorbed water, and the second at ca. 400 °C corresponding to the release of crystallization water. Zirconium and silica oxides offer good thermal stability [48]. As shown in Figure 1, all of the materials prepared in this study have high thermal stability. A mass loss of 20% was observed over the whole analyzed temperature range (30-1000 °C) for ZrO2_Cys ( Figure 1a). An additional mass loss at 900 °C was observed for the ZrO2_Cys_GA system, probably associated with the thermal decomposition of glutaraldehyde. For SiO2_Cys and SiO2_Cys_GA, the mass loss was smaller than in the case of the zirconium materials. In this case the total mass loss was 10% over the analyzed range of temperatures (30-1000 °C). To summarize these results, it was found that the proposed cysteine-functionalized ZrO2 or SiO2 materials are suitable for use as supports in the immobilization of enzymes. Moreover, contents of N, C, H and S were determined to confirm the functionalization with cysteine and glutaraldehyde. The results are given in Table 2. To confirm the effectiveness of the modification, the table also provides data for pure ZrO2 and SiO2. An analysis of these data shows that the modification with cysteine and glutaraldehyde was successful. The contents of N, C and S increased for the sample with Cys and GA. The systems ZrO2_Cys and ZrO2_Cys_GA have higher contents of nitrogen, carbon and sulfur (N = 0.49%, C = 3.32% and S = 2.18%; N = 0.49%, C = 4.46% Moreover, contents of N, C, H and S were determined to confirm the functionalization with cysteine and glutaraldehyde. The results are given in Table 2. To confirm the effectiveness of the modification, the table also provides data for pure ZrO 2 and SiO 2 . An analysis of these data shows that the modification with cysteine and glutaraldehyde was successful. The contents of N, C and S increased for the sample with Cys and GA. The systems ZrO 2 _Cys and ZrO 2 _Cys_GA have higher contents of nitrogen, carbon and sulfur (N = 0.49%, C = 3.32% and S = 2.18%; N = 0.49%, C = 4.46% and S = 2.14%, respectively) than SiO 2 _Cys and SiO 2 _Cys_GA (N = 0.07%, C = 1.01% and S = 0.12%; N = 0.09%, C = 1.57% and S = 0.11%, respectively). These results show that functionalization with Cys and GA was more effective for ZrO 2 -based samples.

Spectroscopic, Porous and Zeta Potential Analysis of Samples before and after Immobilization
FTIR analysis is one of the most common methods used to identify the characteristic groups present in samples. In this study, samples were evaluated by this method both before and after immobilization. The FTIR spectra are shown in Figure 2, and the characteristic groups are summarized in Table 3   Amide I 1624 The porosity of materials also plays an important role in enzyme immobilization. A classical method of immobilization is adsorption. Materials may be potential supports if they have a welldeveloped porous structure. In Table 4, the basic porous parameters (surface area, maximum pore volume and mean pore diameter) are shown. The ZrO2_Cys and ZrO2_Cys_GA systems have large surface areas of 295 and 262 m 2 /g, respectively. After immobilization the surface area decreased,  The porosity of materials also plays an important role in enzyme immobilization. A classical method of immobilization is adsorption. Materials may be potential supports if they have a well-developed porous structure. In Table 4, the basic porous parameters (surface area, maximum pore volume and Processes 2020, 8, 885 9 of 17 mean pore diameter) are shown. The ZrO 2 _Cys and ZrO 2 _Cys_GA systems have large surface areas of 295 and 262 m 2 /g, respectively. After immobilization the surface area decreased, attaining values of 292 m 2 /g for ZrO 2 _Cys_Lac and 146 m 2 /g for ZrO 2 _Cys_GA_Lac. This is probably associated with the attachment of laccase to the support surface and the blocking of pores. The SiO 2 -based materials (SiO 2 _Cys and SiO 2 _Cys_GA) have smaller surface areas (20 and 17 m 2 /g) than the ZrO 2 -based materials. However, the surface area again decreased after immobilization. In this case, the pore diameter was also reduced after immobilization, which may be related to the deposition of laccase inside the pores. In the final part of the physicochemical analysis, the zeta potential (ζ) and isoelectric point (IEP) were determined (Figure 3). Based on the zeta potential values, the electrostatic interactions between the enzyme and support were investigated. The value of ζ for ZrO 2 _Cys at all analyzed pH (2-10) is between 40 and -50 mV, with the isoelectric point at 4.57 (Figure 3a). Small changes in the zeta potential are observed after the immobilization of laccase on ZrO 2 _Cys (ZrO 2 _Cys_Lac), and the isoelectric point of ZrO 2 _Cys_Lac occurs at pH = 4.00. Analyzing the zeta potential and isoelectric point values of ZrO 2 _Cys_GA (before and after immobilization) no significant changes were observed (Figure 3b). A similar situation was found for the samples based on SiO 2 , before and after immobilization of laccase. However, the zeta potential of the proposed biocatalytic systems is more negative. This is associated with the slipping plane, which is slightly stronger after the immobilization of laccase onto cysteine-functionalized ZrO 2 and SiO 2 materials [49,50]. attaining values of 292 m 2 /g for ZrO2_Cys_Lac and 146 m 2 /g for ZrO2_Cys_GA_Lac. This is probably associated with the attachment of laccase to the support surface and the blocking of pores. The SiO2based materials (SiO2_Cys and SiO2_Cys_GA) have smaller surface areas (20 and 17 m 2 /g) than the ZrO2-based materials. However, the surface area again decreased after immobilization. In this case, the pore diameter was also reduced after immobilization, which may be related to the deposition of laccase inside the pores. In the final part of the physicochemical analysis, the zeta potential (ζ) and isoelectric point (IEP) were determined (Figure 3). Based on the zeta potential values, the electrostatic interactions between the enzyme and support were investigated. The value of ζ for ZrO2_Cys at all analyzed pH (2-10) is between 40 and -50 mV, with the isoelectric point at 4.57 (Figure 3a). Small changes in the zeta potential are observed after the immobilization of laccase on ZrO2_Cys (ZrO2_Cys_Lac), and the isoelectric point of ZrO2_Cys_Lac occurs at pH = 4.00. Analyzing the zeta potential and isoelectric point values of ZrO2_Cys_GA (before and after immobilization) no significant changes were observed (Figure 3b). A similar situation was found for the samples based on SiO2, before and after immobilization of laccase. However, the zeta potential of the proposed biocatalytic systems is more negative. This is associated with the slipping plane, which is slightly stronger after the immobilization of laccase onto cysteine-functionalized ZrO2 and SiO2 materials [49,50].  Based on the FTIR, BET and potential zeta results, a mechanism for the immobilization of laccase on cysteine-functionalized MxOy (activated or not with glutaraldehyde) was proposed and presented in Figure 4. The results from FTIR and zeta potential analysis suggest that the immobilization of laccase on MxOy_Cys without glutaraldehyde take place through non-specific forces such as Van der Waals and electrostatic interactions. Whereas, the activation of MxOy_Cys with glutaraldehyde is usually via a first ionic exchange. Then the immobilization by covalent bonds is possible. In addition, Based on the FTIR, BET and potential zeta results, a mechanism for the immobilization of laccase on cysteine-functionalized M x O y (activated or not with glutaraldehyde) was proposed and presented in Figure 4. The results from FTIR and zeta potential analysis suggest that the immobilization of laccase on M x O y _Cys without glutaraldehyde take place through non-specific forces such as Van der Waals and electrostatic interactions. Whereas, the activation of M x O y _Cys with glutaraldehyde is usually via a first ionic exchange. Then the immobilization by covalent bonds is possible. In addition, the surface of cysteine-functionalized M x O y is positively and negatively charged. When that surface is activated with glutaraldehyde, the mixed anion/cationic exchange takes place [26]. On the other hand, the changes in porous structure observed after immobilization suggest that enzyme was adsorbed inside the pores of support.
Processes 2020, 8, x FOR PEER REVIEW 10 of 17 the surface of cysteine-functionalized MxOy is positively and negatively charged. When that surface is activated with glutaraldehyde, the mixed anion/cationic exchange takes place [26]. On the other hand, the changes in porous structure observed after immobilization suggest that enzyme was adsorbed inside the pores of support.

Catalytic Properties of the Obtained Biocatalytic Systems
Catalytic parameters of the prepared biocatalytic systems are presented in Table 5. These data show that the highest quantity of laccase was immobilized on ZrO2_Cys (250 mg per 1 g of support), while for the other systems the quantities of immobilized enzyme were smaller (212-225 mg/g). In all cases, the immobilization yield is in the range 94-99%. The activity of biocatalytic systems determines their possible applications. The specific activity of the ZrO2_Cys_Lac biocatalytic system was found to be 19.3 U/g, compared with 25 U/mg for free laccase. This means that the immobilized laccase (ZrO2_Cys_Lac) retained about 77% of the initial activity. The system ZrO2_Cys_GA_Lac had slightly lower specific activity (13.5 U/mg) and activity retention (ca. 54%). The activity of laccase immobilized on ZrO2_Cys_GA is lower than in the case of ZrO2_Cys, because the modification with glutaraldehyde has an impact on both the enzyme structure and the enzymatic activity. For the other biocatalytic systems, significantly smaller values were obtained (0.6 U/mg and 2.6% for SiO2_Cys_Lac; 7.1 U/mg and 28.5% for SiO2_Cys_GA_Lac). Table 5 also contains kinetic parameters of the obtained biocatalytic systems, which indicate the affinity of laccase to its substrate. The Michaelis constant (KM) for the laccase immobilized on ZrO2-based materials (0.11-0.14 mM) is smaller than the value for free laccase (0.18 mM). Changes are also observed for the maximum retention velocity (Vmax). The Vmax values for laccase immobilized on cysteine-functionalized ZrO2 were higher (0.095 mM/s for ZrO2_Cys_Lac; 0.031 mM/s for ZrO2_Cys_GA_Lac) than the value for free laccase (0.027 mM/s). These results indicate a slightly higher substrate affinity in the case of the immobilized biomolecules. The kinetic parameters of SiO2_Cys_Lac and SiO2_Cys_GA_Lac are similar to those of the free enzyme. Similar results were obtained by Qiu et al. [51], which immobilized laccase onto inorganic mesoporous silica and natural organic polymer like chitosan using functional ionic liquid as bridging agent (SBA-CIL-CS). Kinetic parameters measurement showed that the SBA-CIL-CS-Lac had the outstanding affinity to the substrate.

Catalytic Properties of the Obtained Biocatalytic Systems
Catalytic parameters of the prepared biocatalytic systems are presented in Table 5. These data show that the highest quantity of laccase was immobilized on ZrO 2 _Cys (250 mg per 1 g of support), while for the other systems the quantities of immobilized enzyme were smaller (212-225 mg/g). In all cases, the immobilization yield is in the range 94-99%. The activity of biocatalytic systems determines their possible applications. The specific activity of the ZrO 2 _Cys_Lac biocatalytic system was found to be 19.3 U/g, compared with 25 U/mg for free laccase. This means that the immobilized laccase (ZrO 2 _Cys_Lac) retained about 77% of the initial activity. The system ZrO 2 _Cys_GA_Lac had slightly lower specific activity (13.5 U/mg) and activity retention (ca. 54%). The activity of laccase immobilized on ZrO 2 _Cys_GA is lower than in the case of ZrO 2 _Cys, because the modification with glutaraldehyde has an impact on both the enzyme structure and the enzymatic activity. For the other biocatalytic systems, significantly smaller values were obtained (0.6 U/mg and 2.6% for SiO 2 _Cys_Lac; 7.1 U/mg and 28.5% for SiO 2 _Cys_GA_Lac). Table 5 also contains kinetic parameters of the obtained biocatalytic systems, which indicate the affinity of laccase to its substrate. The Michaelis constant (K M ) for the laccase immobilized on ZrO 2 -based materials (0.11-0.14 mM) is smaller than the value for free laccase (0.18 mM). Changes are also observed for the maximum retention velocity (V max ). The V max values for laccase immobilized on cysteine-functionalized ZrO 2 were higher (0.095 mM/s for ZrO 2 _Cys_Lac; 0.031 mM/s for ZrO 2 _Cys_GA_Lac) than the value for free laccase (0.027 mM/s). These results indicate a slightly higher substrate affinity in the case of the immobilized biomolecules. The kinetic parameters of SiO 2 _Cys_Lac and SiO 2 _Cys_GA_Lac are similar to those of the free enzyme. Similar results were obtained by Qiu et al. [51], which immobilized laccase onto inorganic mesoporous silica and natural organic polymer like chitosan using functional ionic liquid as bridging agent (SBA-CIL-CS). Kinetic parameters measurement showed that the SBA-CIL-CS-Lac had the outstanding affinity to the substrate.
The values of catalytic properties show that the kinetic parameters are changed after the immobilization process. The changes in kinetic parameters are caused by the transformations of the protein structure and the immobilization methods. Furthermore, a decrease in K M leads to an increase in the enzyme's affinity to the substrate. This probably occurs when the electric charges on the support and substrate are of different sign [52,53]. The immobilized enzymes are less sensitive to pH and temperature changes, retain high activity after many days of storage, and can be used in several reaction cycles. Figure 5 shows the influence of pH, temperature, storage stability and reusability on the activity of free and immobilized laccase. All of the biocatalytic systems obtained in this study retain good activity under various chemical and thermal conditions. Furthermore, good activity is retained after a number of days of storage and after several reaction cycles. The ZrO 2 _Cys_Lac and ZrO 2 _Cys_GA_Lac systems maintain residual activity above 40% at all analyzed pH and temperature values. Furthermore, ZrO 2 _Cys_Lac retains high (above 90%) activity after 30 days of storage and after 10 reaction cycles. Laccase immobilized on SiO 2 _Cys_GA exhibits slightly lower activity (above 60%) in the same conditions. It preserves about 20-30% of its initial activity at pH = 3-7 and ca. 40% at temperatures of 30-70 • C. Its activity after 30 days of storage and after 10 reaction cycles is only 30-40% and 50-60%, respectively. Table 6 summarizes the above results together with the results of other studies in which different cysteine-functionalized supports were used to immobilize various enzymes. The table shows that the results obtained using cysteine-functionalized ZrO 2 and SiO 2 materials as supports for laccase are satisfactory compared with the other results. As shown, cysteine has been used to modify Ag, Cu, ZnO and poly(glycidyl methacrylate)-SiO 2 , utilized as supports for alkaline phosphatase, urease and lipase, respectively [15,16,18,19]. Upadhyay et al. [15] proposed a cysteine-Ag/AP biocatalytic system, which exhibited a specific activity of 6.31 U/mg and activity retention of 67%, and retained 60% of its initial activity after seven reaction cycles. Kumar et al. [16] and Verma et al. [18] immobilized urease on cysteine-Cu and cysteine-ZnO, respectively. In both cases the activity retention was ca. 72%. Good results were also obtained by Chen et al. [19], who prepared cysteine-poly(glycidyl methacrylate)-SiO 2 /Lip biocatalysts with high specific activity (44.1 U/mg) and activity retention (63.3%). That system was also used over eight reaction cycles, retaining 40% of its activity.
Summarizing performed research it has been shown, that the catalytic properties of enzyme in different pH, temperature, storage stability and reusability were improved. The support stabilizes and stiffness the enzyme structure, which consequently protect enzyme against denaturation in extreme pH and temperature conditions. Moreover, the carrier exposes the active sites of the catalyst for easy attachment of substrate molecules and reduce diffusional resistance of the substrates and products. The best catalytic properties were obtained when laccase was immobilized onto ZrO 2 _Cys. It can be associated with the properties of that materials, especially well-developed porous structure. Furthermore, the data from elemental analysis showed that the cysteine was successfully introduced into the ZrO 2 . The activation with glutaraldehyde causes lowering of catalytic activity. It may be related with the presence of covalent bonds, which can block active site of enzyme and in consequently reduced enzymatic activity. (c) (d) Figure 5. Influence of (a) pH, (b) temperature, (c) storage stability and (d) reusability on relative activity of free and immobilized laccase. Table 6 summarizes the above results together with the results of other studies in which different cysteine-functionalized supports were used to immobilize various enzymes. The table shows that the results obtained using cysteine-functionalized ZrO2 and SiO2 materials as supports for laccase are satisfactory compared with the other results. As shown, cysteine has been used to modify Ag, Cu, ZnO and poly(glycidyl methacrylate)-SiO2, utilized as supports for alkaline phosphatase, urease and lipase, respectively [15,16,18,19]. Upadhyay et al. [15] proposed a cysteine-Ag/AP biocatalytic system, which exhibited a specific activity of 6.31 U/mg and activity retention of 67%, and retained 60% of its initial activity after seven reaction cycles. Kumar et al. [16] and Verma et al. [18] immobilized urease on cysteine-Cu and cysteine-ZnO, respectively. In both cases the activity retention was ca. 72%. Good results were also obtained by Chen et al. [19], who prepared cysteine-poly(glycidyl methacrylate)-SiO2/Lip biocatalysts with high specific activity (44.1 U/mg) and activity retention (63.3%). That system was also used over eight reaction cycles, retaining 40% of its activity.
Summarizing performed research it has been shown, that the catalytic properties of enzyme in different pH, temperature, storage stability and reusability were improved. The support stabilizes and stiffness the enzyme structure, which consequently protect enzyme against denaturation in extreme pH and temperature conditions. Moreover, the carrier exposes the active sites of the catalyst for easy attachment of substrate molecules and reduce diffusional resistance of the substrates and products. The best catalytic properties were obtained when laccase was immobilized onto ZrO2_Cys. It can be associated with the properties of that materials, especially well-developed porous structure. Furthermore, the data from elemental analysis showed that the cysteine was successfully introduced into the ZrO2. The activation with glutaraldehyde causes lowering of catalytic activity. It may be related with the presence of covalent bonds, which can block active site of enzyme and in consequently reduced enzymatic activity.

Decolorization of Alizarin Red S
Laccase is an enzyme which is capable of degrading and decolorizing organic dyes from wastewaters. In this study, the obtained biocatalytic systems were used for the decolorization  Figure 6, including the effect of time, pH and temperature. As shown in Figure 6a, the ZrO 2 -based biocatalytic systems (ZrO 2 _Cys_Lac and ZrO 2 _Cys_GA_Lac) produced a higher efficiency of decolorization of ARS dye over the analyzed process duration, as compared with the SiO 2 -based biocatalytic systems (SiO 2 _Cys_Lac and SiO 2 _Cys_GA_Lac). The highest decolorization efficiency was achieved after 24 h (95% for ZrO 2 _Cys_Lac and 85% for ZrO 2 _Cys_GA_Lac), but an efficiency of ca. 90% was already reached after 5 h. After the same time, the efficiency of decolorization of ARS using SiO 2 _Cys_Lac and SiO 2 _Cys_GA_Lac reached 35% and 20%, respectively. Laccase immobilized on ZrO 2 -based materials results in higher decolorization of ARS dye because of the high specific activity and activity retention of these systems. It was observed that Alizarin Red S can be successfully decolorized by immobilizing laccase on ZrO 2 -based materials. Furthermore, the efficiency of decolorization of Alizarin Red S in this study was higher than in previous reports where immobilized laccase was used. Zhao et al. [54] immobilized laccase on mesostructured cellular foam silica (MCF), and obtained maximum decolorization of Alizarin Red S equal to 73%. Similarly, Lu et al. [55] used alginate-chitosan microcapsules as a support for laccase. That biocatalytic system was utilized in the decolorization of ARS, and the efficiency of decolorization was measured at 70%.

Decolorization of Alizarin Red S
Laccase is an enzyme which is capable of degrading and decolorizing organic dyes from wastewaters. In this study, the obtained biocatalytic systems were used for the decolorization of Alizarin Red S (ARS) dye. The results are presented in Figure 6, including the effect of time, pH and temperature. As shown in Figure 6a, the ZrO2-based biocatalytic systems (ZrO2_Cys_Lac and ZrO2_Cys_GA_Lac) produced a higher efficiency of decolorization of ARS dye over the analyzed process duration, as compared with the SiO2-based biocatalytic systems (SiO2_Cys_Lac and SiO2_Cys_GA_Lac). The highest decolorization efficiency was achieved after 24 h (95% for ZrO2_Cys_Lac and 85% for ZrO2_Cys_GA_Lac), but an efficiency of ca. 90% was already reached after 5 h. After the same time, the efficiency of decolorization of ARS using SiO2_Cys_Lac and SiO2_Cys_GA_Lac reached 35% and 20%, respectively. Laccase immobilized on ZrO2-based materials results in higher decolorization of ARS dye because of the high specific activity and activity retention of these systems. It was observed that Alizarin Red S can be successfully decolorized by immobilizing laccase on ZrO2-based materials. Furthermore, the efficiency of decolorization of Alizarin Red S in this study was higher than in previous reports where immobilized laccase was used. Zhao et al. [54] immobilized laccase on mesostructured cellular foam silica (MCF), and obtained maximum decolorization of Alizarin Red S equal to 73%. Similarly, Lu et al. [55] used alginate-chitosan microcapsules as a support for laccase. That biocatalytic system was utilized in the decolorization of ARS, and the efficiency of decolorization was measured at 70%. The immobilization process serves to produce biocatalytic systems that can be used in a range of reaction conditions. Therefore, decolorization was carried out under different pH and temperature conditions (Figure 6b,c). Laccase immobilized on ZrO2_Cys and ZrO2_Cys_GA achieved a high efficiency of decolorization (above 70%) in the whole of the analyzed pH and temperature ranges. Laccase immobilized on SiO2_Cys and SiO2_Cys_GA resulted in significantly lower efficiency of decolorization of ARS dye, reaching at most 40%. The results indicate that the biocatalytic systems proposed in this study (especially ZrO2_Cys_Lac and ZrO2_Cys_Ga_Lac) have potential applications in the decolorization of dyes from wastewaters. The immobilization process serves to produce biocatalytic systems that can be used in a range of reaction conditions. Therefore, decolorization was carried out under different pH and temperature conditions (Figure 6b,c). Laccase immobilized on ZrO 2 _Cys and ZrO 2 _Cys_GA achieved a high efficiency of decolorization (above 70%) in the whole of the analyzed pH and temperature ranges. Laccase immobilized on SiO 2 _Cys and SiO 2 _Cys_GA resulted in significantly lower efficiency of decolorization of ARS dye, reaching at most 40%. The results indicate that the biocatalytic systems proposed in this study (especially ZrO 2 _Cys_Lac and ZrO 2 _Cys_Ga_Lac) have potential applications in the decolorization of dyes from wastewaters.

Conclusions
The experiments performed in this study showed that large quantities of enzyme were attached to the proposed materials. Cysteine-functionalized ZrO 2 produced a higher immobilization yield and enzymatic activity than cysteine-functionalized SiO 2 , which may suggest the superior ability of ZrO 2 -based materials to attach to enzymes. Good thermal and electrokinetic stability, a well-developed porous structure and the presence of specific groups enable the use of M x O y -based materials for the immobilization of laccase from Trametes versicolor. In addition, based on that analysis the catalytic (A S and A R ) and kinetic (K M and V max ) parameters of the obtained biocatalytic systems were determined based on the oxidation of ABTS. The values of these parameters indicate the higher affinity of immobilized laccase to the substrate compared with the free enzyme. Moreover, laccase immobilized on ZrO 2 -based support retains high relative activity over a wide range of pH (>40%) and temperature (>50%), and also after 30 days of storage (>60%) and 10 reaction cycles (>60%). Most importantly, very good results were achieved in the decolorization of Alizarin Red S. In these tests, a high efficiency of decolorization (97%) was obtained. The biocatalytic systems proposed in this study, based on cysteine-functionalized metal oxide, may be used in removing other organic and pharmaceutical pollutants from wastewaters.