1. 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 synthesis, metabolism, and detoxification. Due to its thiol groups, cysteine can easily be oxidized to cystine, a dimeric amino acid. This reaction is reversible and allows the control of a wide range of biological activities and protein structures, and, therefore, the determination of cysteine in biological matrices and pharmaceutical preparation is highly important [
5]. The cysteine is using in food chemistry as a reducing agent in production of French bread, crackers and cookies. The cysteine is effective in preventing browning of fruit juice during concentration. They also prevent the development of off-flavor in stored orange juice. In addition, in flavor chemistry, cysteine is an important source of sulfur in a variety of aromas [
6].
Metal oxides (M
xO
y: SiO
2, ZrO
2, ZnO, Fe
2O
3, Al
2O
3, etc.) are commonly used as supports for the immobilization of enzymes because of their good thermal and chemical stability, in addition to excellent mechanical resistance [
7,
8,
9,
10]. These materials are easy to synthesize, which makes them relatively cheap. Moreover, the surface of M
xO
y particles can be modified by various groups, enabling the attachment of enzymes to the surface. The thiol groups present in cysteine have a strong tendency to be adsorbed onto the surfaces of certain metals [
11]. According to reports in the literature, cysteine has been used for modifying nanoparticles of gold [
12,
13], silver [
14,
15], copper [
16] and nickel [
17]. Cysteine is also used in the synthesis or modification of nanoparticles which are then used as supports for enzymes. For example, Verma et al. [
18] synthesized ZnO using
l-cysteine, and then immobilized urease on the cysteine/ZnO nanoparticles. Results have also been reported concerning the modification of silica with cysteine and the use of the obtained material for immobilization of lipase [
19]. Magnetic nanoparticles have also been functionalized with
Cys and used as a support for xylose reductase [
20]. Bezbradica et al. [
21] prepared a matrix by chemical activation with cysteine and glutaraldehyde. The proposed support was used to immobilize four different molecules (trypsin, penicillin acylase G, lipase, and
E. coli BL21 cell extract). In these studies, the immobilization is promoted through a two-step mechanism: in a first step, the enzyme is adsorbed on the support via an anionic exchange mechanism and, then, the covalent immobilization occurs. Immobilization on standard amino support activated with glutaraldehyde is usually via a first ionic exchange, then the covalent bonds may be produced. This is, in fact, a heterofunctional support [
22,
23]. In most cases, the imine linkage is formulated between glutaraldehyde-activated support and amino groups of enzyme, which should be later reduced to strengthen the linkage. However, most of the proteins are immobilized at neutral pH on glutaraldehyde-activated supports because imine in aqueous medium is unstable and the equilibrium enzyme support is shifted to the dissociated form. According to this, the linkages on glutaraldehyde-activated supports are performed through the reaction with cycled forms of the glutaraldehyde. This may cause that linkages are more stable than the imines [
24,
25]. Additionally, the surface of the support functionalized with cysteine is positively and negatively charged. If that surface is activated with glutaraldehyde, the mixed anionic/cationic exchange between enzyme and support takes place [
26]. However, it also should be mentioned that, if the support has primary amino groups, and is modified with glutaraldehyde, the covalent bonds can also take place [
27,
28].
Dyes are organic, colored compounds which are capable of dyeing animal fibers (wool, silk), plant fibers (cotton, flax), leather, etc. The color of organic dyes depends on the presence in the molecule of chromophores (responsible for color formation) and auxochromes (electron donors which also increase the color by improving the solubility and adhesion of the dye to the fiber) [
29,
30]. Organic dyes are classified as chemical (e.g., nitro, anthraquinone, indigoid) and technical (e.g., acid, basic, vat, and reactive) [
31]. Organic dyes are among the most significant contaminants of wastewater, because of their extensive use in numerous industries [
32]. Alizarin Red S (ARS) is a 3-substituted derivative of 1,2-dihydroxy-9,10-anthraquinone, which belongs to the group of most durable dyes in textile wastewaters. That dye is water soluble and has application in histological studies to identifying calcium in tissues and vital staining of bone. The ARS can induce structural and functional changes to serum albumins [
33]. The research show that ARS are also introduced adverse effects to organisms, such as oxidative damages. Moreover, other experimental show that anthraquinone and its sulfonated 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 MxOy material as a support for enzyme immobilization. The materials used are SiO2 and ZrO2, 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 MxOy 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).
2. Materials and Methods
2.1. Materials
Sol-gel method: tetraethyl orthosilicate (TEOS), zirconium isopropoxide (ZIP), NH3aq (25%), ethanol, isopropanol, l-cysteine (Cys), HCl. Immobilization process: glutaraldehyde (GA), laccase from Trametes versicolor (Lac), buffers: acetate (0.1 M; pH = 2–5), phosphate (0.1 M; pH = 6–8) and TRIS (0.1 M; pH = 9–10), Bradford reagent, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Decolorization of dyes: Alizarin Red S (ARS). All materials were purchased from Sigma-Aldrich® (St. Louis, MO, USA).
2.2. Synthesis of l-Cysteine-Functionalized MxOy
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, SiO2_Cys and ZrO2_Cys were activated with glutaraldehyde (5% in pH = 7 buffer) for 24 h. This led to the systems SiO2_Cys_GA and ZrO2_Cys_GA.
The four kind of supports (SiO2_Cys; SiO2_Cys_GA, ZrO2_Cys and ZrO2_Cys_GA) were used in the next step of research.
2.3. 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.
2.4. Physicochemical Characterization
Spectroscopic, thermogravimetric, porous structure, zeta potential and elemental analysis were applied to characterize the samples obtained during the study.
The pure supports (ZrO2_Cys, ZrO2_Cys_GA, SiO2_Cys and SiO2_Cys_GA) were evaluated by means of thermal and elemental analysis. Thermogravimetric analysis (TG/DTG) was performed using a Jupiter STA 449F3 thermogravimetric analyzer (Netzsch, Selb, Germany). A sample (ca. 5 mg) was heated in a nitrogen atmosphere in the temperature range 30–1000 °C.
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 N2 sorption the analysis was carried out. Using the BET (Brunauer–Emmett–Teller) and BJH (Barrett–Joyner–Halenda) methods, the surface area (ABET), total pore volume (Vp) and mean pore diameter (Sp) were assessed. Due to the high accuracy of the instrument used, surface area was determined to an accuracy of 0.1 m2/g, pore volume to 0.01 cm3/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.
2.5. 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
AR—retention activity (%);
AS—specific activity (U/mg
enzyme);
AS1—specific activity of immobilized Lac (U/mg);
AS0—specific activity of free Lac (U/mg);
AAp—apparent activity (U/g
support);
P—amount of immobilized Lac (mg/g).
Additionally, the kinetic parameters (KM, the Michaelis-Menten constant; and Vmax, 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.
2.6. 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;
CB and
CA are the concentrations of ARS dye before and after the decolorization process, respectively.