Introduction

Laccases (E.C. 1.10.3.2) are multicopper-containing enzymes that catalyze the one-electron oxidation of various phenolic and non-phenolic compounds using the simultaneous reduction of an oxygen molecule to water. According to several reports, laccase has recently gained a significant role in many industrial, environmental, diagnostic, bioremediation, and biofuel cell applications [4, 5, 8, 12, 14, 26]. This increased utilization of laccase requires industrial-scale processes to increase cost effectiveness. The free enzyme readily loses much of its activity under different sets of conditions and in the presence of inhibitors. To overcome such problems, immobilization of the enzyme has been widely demonstrated on different kinds of support materials, which has provided significant improvement in its properties [8]. In general, however, the results obtained with the immobilized enzyme have been highly unpredictable with respect to both yield and efficiency owing to variations in the properties of the enzyme and its support materials under different immobilization conditions [14, 27]. Immobilization of enzymes is primarily based on physical, covalent, and affinity interactions. Among the different methods employed, covalent immobilization has recently become more popular owing to its specificity, stability, and rapidity [6, 17-20, 25].

Immobilization of laccase has been reported using a variety of support materials, including alginate beads [11], magnetic-chitosan [2], and Amberlite [21]. In recent studies, nanocarriers are used most often as support materials for immobilization of the enzyme because of their unique properties, such as high surface area, shape retention, availability in different sizes and compositions, and characteristics after functional activation of the surface, even when very harsh chemical modifications are carried out [1, 14, 22]. Several methods have been developed for immobilization of the enzyme, but robust support materials and immobilization methods are still being sought to overcome problems associated with industrial applications of the pure enzyme.

Successful development of an immobilized enzyme system depends on the type of support material, the properties of the enzyme, and the immobilization process. For support materials, properties such as morphology, composition, hydrophobicity, particle size, specific surface area, functional surface group, and rigidity are critical factors [9, 19, 25]. Immobilization yield (IY) and immobilization efficiency (IE) are significantly influenced by the support material, and optimization of the immobilization conditions is critical for success. Among the different types of support matrix, silica-based nanoparticles are highly suitable for the immobilization of enzyme owing to their properties, such as environmental benignness, high biocompatibility, and resistance towards organic solvents and microbial attacks. In the present study, immobilization of laccase was performed on functionally activated SiO2 nanoparticles to achieve high stability and reusability. We studied the optimization of the following process parameters: temperature, pH, thermal stability, kinetic parameters of free and immobilized enzyme, and reusability. The results obtained in this work show that immobilized laccase is very efficient and stable compared with the free enzyme, and has high reusability. This efficient immobilized system has promise for industrial applications.

Materials and Methods

Materials

Unless otherwise stated, all chemicals and reagents used in the experiments were of analytical grade from commercial sources. SiO2 nanoparticles were purchased from Nanostructured and Amorphous Materials, Inc. (Houston, TX, USA). Laccase from Trametes versicolor, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and glutaraldehyde (50% in water) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Functional Activation of SiO2 Nanoparticles

The modification of silicon nanoparticles to produce aldehyde groups was conducted based on a previously reported method [13]. The typical protocol was as follows: nanoparticles (1 g) were sonicated for 30 min in distilled water, and the particles were collected by centrifugation and resuspended in 1 M glutaraldehyde for 2 h at room temperature. Afterward, the modified particles were washed five times with distilled water and separated via centrifugation (4,000 ×g) for 30 min.

To modify the nanoparticles to have cyanogen groups, the sonicated and washed nanoparticles were collected by centrifugation, added to 600 μl of 2 M sodium carbonate, and the slurry was cooled to 0℃. Next, 160 μl of 0.45 g/ml CNBr dissolved in dimethylformamide was added, mixed vigorously for 2 min, and incubated for 20 min on a shaker. The activated support material was then washed with 5 volumes of cold distilled water [25].

The modification of nanoparticles to give carbodiimide groups was carried out as follows: the sonicated and washed particles were suspended in 10 ml of 0.1 M sodium acetate buffer (pH 4.5). Then, 200 mg of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene-sulfonate was added to the support slurry. This was mixed for 4 h at room temperature, and the particles were washed with 500 ml of cold 0.1 M sodium phosphate buffer (pH 7.0) [25].

Immobilization of Laccase

The modified carrier (10 mg) was mixed with 1 ml of laccase (0.5 mg protein) and incubated for 24 h at 4℃ with shaking at 150 rpm. After immobilization, the beads were collected by centrifugation and washed three times with 100 mM sodium acetate buffer (pH 5.0). The protein concentration of the washed solution was measured using the Bradford method [3], and the activities of the immobilized laccase were determined. The IE and IY were calculated as follows: IE = 100 × (αi/αf) and IY = 100 × [(ρi-ρw-ρs)/ρi], where αi is the total activity of the immobilized enzyme and αf is the total activity of the free enzyme; ρi is the total protein content of the crude enzyme preparation, and ρw and ρs are the protein concentrations of the wash solution and supernatant after immobilization, respectively [25]. All assays were performed in triplicate.

Enzyme Assay

Laccase activity was determined spectrophotometrically (Varian Cary 100 Bio UV–Vis spectrophotometer; Palo Alto, CA, USA), using ABTS as the substrate. The oxidation of ABTS was observed by measuring absorbance increases at 420 nm (εmax = 3.6 × 104/M × cm). Enzyme activity was expressed as international units (IU), where 1 IU represents the amount of enzyme that forms 1 μmol of product per minute under standard assay conditions [10, 12].

Optimization of Immobilization Conditions

The effect of pH on laccase immobilization was assessed over the range of pH 2–7 in the following buffers (100 mM): glycine-HCl (pH 2–3), sodium acetate (pH 4–6), and sodium phosphate (pH 7). Similarly, immobilization temperature optimization was carried out at temperatures of 4℃, 16℃, 25℃, and 30℃. To determine the optimum loading on the SiO2 nanoparticles, 25, 50, 100, 150, or 200 mg of laccase protein was added to 1 g of support. Finally, incubation periods of 16, 24, 36, and 48 h were tested. All assays were performed in triplicate. The optimization data are presented as the averages of statistically relevant measurements with their associated standard deviations.

Characterization of Immobilized Laccase

The effects of pH and temperature on free and immobilized laccase activities were measured by monitoring the oxidation of ABTS in different buffers (50 mM). The pH effect was determined in buffered solutions varying from pH 2–3 (glycine-HCl), 3–4 (sodium citrate), 4–6 (sodium acetate), and 6–7 (sodium phosphate). The thermal effect was determined by carrying out enzyme assays at 25–60℃ in different buffers (50 mM) at the optimum pH.

The kinetic parameters of free and immobilized laccase were determined at 25℃ using ABTS concentrations in the range 0.005– 2.0 mM in 50 mM sodium citrate buffer at the optimum pH. Kinetic parameters (apparent Km and Vmax) for substrates were obtained using nonlinear regression-fitting analyses of the data in GraphPad Prism 5 software (Grappa Software, Inc.; CA USA). All assays were performed in triplicate. The kinetic data are presented as the averages of statistically relevant measurements with their associated standard deviations.

Thermal Stability and Determination of Thermal Deactivation Constant

Thermal stabilities were determined by incubating free and immobilized laccases from 2 h to 4 days at temperatures ranging from 30℃ to 60℃ in sodium citrate buffer (50 mM) at the optimum pH. The thermal denaturation kinetics of free and immobilized laccases were determined using reaction kinetic analysis for a general first-order rate of reaction derived from the Arrhenius Eq. (1):

where Ao is the initial laccase activity, A is the residual laccase activity after incubation at a particular temperature, kd is the rate constant (per h), and t is duration of incubation (h) [7].

The half-life (t1/2) of laccase at different temperatures was determined using Eq. (2):

Reusability of Immobilized Laccase

The reusability of laccase immobilized on SiO2 nanoparticles was measured in sodium citrate buffer (50 mM; pH 3.5) at 25℃ over 10 cycles. After each oxidation cycle, the immobilized enzyme was removed by centrifugation at 4,000 ×g for 15 min. The immobilized enzyme was collected and washed with deionized water and buffer. In running the second and subsequent cycles, the immobilized enzyme was resuspended in fresh buffer, added to unoxidized ABTS, and processed as described above. The activity of the immobilized enzyme was considered to be 100% in the initial (zero) cycle. Each cycle is defined here as the complete oxidation of the substrate present in a reaction mixture. All assays were performed in triplicate.

Results

Effect of Functional Group on the Immobilization of Laccase

The SiO2 was modified with glutaraldehyde, 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, or CNBr to give, respectively, aldehyde, carbodiimide, or cyanogen groups on the surface of the nanoparticles. These are the common functional groups used for immobilization via the enzyme’s free amino, carboxyl, or amino groups, respectively. The IY and IE were compared for laccase immobilized on these modified SiO2 nanoparticles, and the results are shown in Fig. 1. The IYs of laccase were 73.2%, 64.5%, and 56.4% with the aldehyde, carbodiimide, and cyanogen groups, respectively. These values are very high in comparison with laccase adsorption onto SiO2 nanoparticles with no functional group modification (approximately 15%). The IEs of laccase on SiO2 nanoparticles modified with the aldehyde, carbodiimide, and cyanogen functional groups were 78.4%, 64.2%, and 61.4%, respectively. The IY and IE were higher for the aldehyde groups than for the carbodiimide and cyanogen groups. Therefore, aldehydegroup-activated SiO2 nanoparticles were selected for further study.

Fig. 1.Effect of functional groups of SiO2 nanoparticles on immobilization yields and efficiency. Black and gray bars represent immobilization yield (%) and efficiency (%), respectively. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Optimization of the Immobilization Conditions

The pH, temperature, enzyme loading, and incubation period were optimized (Fig. 2). The optimum pH for immobilization was 5 in 100 mM sodium acetate buffer (Fig. 2A). A decrease in IY was observed when pH was either lowered from 5 to 2 or raised from 5 to 7. The highest IY (73.2%) was observed at pH 5.0. The IE increased from 10.2% to 78.4% as the pH was raised from 2 to 5, and fell to 62.5% at pH 7. The IE decreased from 78.4% to 34.2% as the temperature was increased from 4℃ to 30℃ (Fig. 2B). Although an increase in IY was observed at higher temperature, a lower immobilization temperature was preferable, as this gave a high IE. The ratio of enzyme to support material (loading) is also a crucial immobilization parameter for achieving maximum IY along with high efficiency. Of the ratios tested (25, 50, 100, 150, or 200 mg laccase protein to 1 g of support), the optimum was 100 mg laccase protein/g of SiO2 nanoparticles (Fig. 2C), which achieved the highest IY (74.1%) and IE (92.9%). For this enzyme-to-support ratio, the IY increased from 58.8% to 75.8% as the incubation period was increased from 16 to 48 h (Fig. 2D). An IE of 92.9% was observed after 36 h of incubation, decreasing thereafter to 84.2% at 48 h.

Fig. 2.Effects of pH (A), temperature (B), ratio of enzyme to support (C), and incubation period (D) on immobilization of laccase on SiO2 nanoparticles functionally activated with glutaraldehyde. Filled and open circles represent immobilization yield (%) and efficiency (%), respectively. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Characterization of the Immobilized Laccase

The characteristics of laccase immobilized under optimum conditions were compared with those of free laccase. The specific activity of free enzyme was 1,440 U/mg of protein (100%) at pH 3.0, and the activity after immobilization was 1,370 U/mg protein (100%) at pH 3.5 (Fig. 3). Increasing the pH to 7 resulted in a reduction of enzyme activities by 0.3% and 5.2% for free and immobilized laccases, respectively. In comparison with free enzyme, immobilized laccase retained high residual activities of 84.7%, 66.8%, and 30.1% at pH 4, 5, and 6, whereas those of the free enzyme were only 59.1%, 33.0%, and 7.2% at the respective pHs. These results suggest that the immobilized enzyme is more resistant to pH changes over a wide range.

After immobilization of T. versicolor laccase on SiO2 nanoparticles, the optimal temperature shifted from 40℃ to 45℃ (Fig. 4) for the maximum residual activity. Further increases in temperature led to decreased residual activity for both the free and immobilized laccases. The residual enzyme activities at 50℃ and 60℃ were 78.5% and 52.5% for the immobilized enzyme, and 37.8% and 32.2% for the free laccase, respectively. The decrease in residual activity of the free enzyme was much higher than that of immobilized laccase. The shift in optimum pH and temperature of the immobilized enzyme may be primarily due to changes in enzyme conformation caused by its binding to the support material. The residual activity of immobilized laccase at pH 6 was improved by approximately 4-fold compared with the free enzyme. Similarity, the residual activity of immobilized laccase at 50℃ and 60℃ was approximately 2-fold higher than that of the free enzyme.

Fig. 3.Effect of pH on the activity of free laccase and on the enzyme immobilized on SiO2 nanoparticles modified with glutaraldehyde. The effect of pH was determined using different buffers (50 mM) to obtain the desired pH in the standard assay. Symbols used for the buffers: circles, glycine-HCl (pH 2.0–3.0); triangles, sodium citrate (pH 3.0–4.0); squares, sodium acetate (pH 4.0–6.0); and diamonds, sodium phosphate (pH 6.0–7.0). Filled and open symbols represent the free and immobilized laccases, respectively. The maximum activities of free and immobilized laccases are shown as 100% residual activity. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

The Michaelis–Menten model was applied to the analysis of kinetic parameters, using ABTS as substrate in 50 mM sodium citrate buffer at 25℃ and at the pH optima of 3.0 and 3.5 for free and immobilized laccases, respectively (Fig. 5). The apparent Vmax and Km values of free laccase were 1,890 μmol/min/mg protein and 29.3 μM. After immobilization, the Km value increased to 46.5 μM and Vmax was slightly reduced to 1,630 μmol/min/mg protein (Table 1).

Fig. 4.Effect of temperature on the activity of free laccase and on the enzyme immobilized on SiO2 nanoparticles. Enzyme activity was measured at various temperatures in sodium citrate buffer (50 mM) under standard conditions at pH 3.0 and 3.5 for free and immobilized laccases, respectively. Filled and open circles represent free and immobilized laccases, respectively. The maximum activities of free and immobilized laccases are shown as 100% residual activity. Data represent the mean of triplicate measurements that varied from the mean by no more than 10%.

Fig. 5.Effect of ABTS concentration on the activity of free laccase and on the enzyme immobilized on SiO2 nanoparticles. Enzyme activity was determined in the presence of ABTS at concentrations in the 0.01–2.00 mM range in sodium citrate buffer (50mM) under standard conditions, at pH 3.0 and 3.5 for free and immobilized laccases, respectively. Filled circles and squares represent free and immobilized laccases, respectively. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Table 1.Kinetic parameters Km and Vmax for immobilized and free laccases.

Thermostability and Reusability

The thermal stability of free and immobilized laccases was evaluated following incubation at different temperatures (Fig. S1). The thermostability of laccase was improved on the glutaraldehyde-modified SiO2 nanoparticles. This suggests that the exposed amine groups on the surface of laccase are readily coupled with aldehyde groups on the nanoparticles to form a stable imine bond that stabilizes the enzyme. The thermal deactivation constant (kd) of the immobilized laccase (0.02) was ~18-fold lower than that of the free enzyme (Table 2). This indicates that the immobilized enzyme is very stable compared with the free enzyme. The t1/2 is the incubation period at which enzymes lose 50% of their activity under optimum conditions. The t1/2 values of the immobilized laccase were 76.9, 64.8, 40.3, 24.6, and 4.0 h at 30℃, 40℃, 45℃, 50℃, and 60℃, which were, respectively, 8.3, 8.8, 10.9, 11.2, and 18.1 times higher than those of the free laccase (Fig. S1). The reusability of immobilized laccase was investigated, as shown in Fig. 6. Immobilized laccase had residual activities of 93.7% and 82.5% after 5 and 10 reaction cycles, respectively.

Table 2.aEach value represents the mean of triplicate measurements and varies from the mean by not more than 10%.

Fig. 6.Reusability of immobilized laccase. One cycle is defined as the time required to oxidize all the ABTS substrate present in the reaction mixture at 25℃ under standard assay conditions. The maximum activity is shown as 100% residual activity. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Discussion

The most important properties of an immobilized system are its kinetics, stability, and reusability. In this work, to achieve optimal efficiency and reusability of laccase immobilized on SiO2 nanoparticles, we evaluated these key properties. First, changing the functional groups (glutaraldehyde, carbodiimide, and cyanogen) for immobilization on the SiO2 nanoparticles produced IYs and IEs in the 56.4-73.2% and 61.4-78.4% ranges, respectively. This variation may be due to differential binding affinities between the functional groups on nanoparticles and the exposed amino acids on laccase. Glutaraldehyde was best for efficient laccase immobilization under the given conditions. The optimum immobilization conditions were pH 5.0, 4℃, an enzyme loading of 100 mg/g support, and 36 h of incubation. The immobilization efficiency obtained in this study is higher than those reported previously (Table 3). For immobilization with glutaraldehyde, the enzyme interacts with the support through ionic exchange, followed by covalent bond formation between the basic amino acid, lysine, and the glutaraldehyde group of the support material [15]. An increase in IYs was observed with increasing temperatures, but IE was reduced from 74.8% at 4℃ to 65.8%, 52.1%, and 34.2% at 16℃, 25℃, and 30℃, respectively. The lower temperature was necessary to achieve high efficiency. The optimum ratio of enzyme to support material depends on their properties. It is likely that nanoparticles with a high specific surface area have high laccase loading capacity. Although a high adsorption of laccase (491 mg/g of support) was observed on magnetic mesoporous silica, the actual immobilization of laccase protein (IY%) was only 19.6% [14]. Under optimum immobilization conditions, we have observed IY of 75.8%. This was about 3.9 times higher than that previous report. The immobilization efficiency (IE%, 92.9%) was also higher.

Table 3.aSupport materials: SNPs - SiO2 nanoparticles; MSNPs - Magnetic SiO2 nanoparticles. bSpecific surface area (m2/g). cIncubation period (h). dImmobilization yield. eImmobilization efficiency. fResidual activity after 10 cycles. gNot applicable.

After enzyme immobilization, there is generally a shift in the optimum conditions due to the interactions between the support and the enzyme, which leads to a change in conformation, thereby reducing enzyme activity in many cases. The optimum pH and temperature of the immobilized laccase were shifted from 3 to 3.5, and from 40℃ to 45℃.The maximum activity of the immobilized laccase at the optimum pH (3.5) was 89% of the maximum activity of the free laccase at pH 3.0. The residual activity of the immobilized enzyme was very high (80–95%) over the 3.5–7.0 pH range as compared with free laccase (20–50%). After immobilization, an improvement in pH stability was observed: the immobilized enzyme retained 2-, 6-, and 17-fold higher residual activities than the free enzyme at pH 5.0, 6.0, and 7.0, respectively. This may be primarily due to interactions between the support and the enzyme. A similar effect was observed with temperature: the immobilized laccase was quite stable at 50℃ and 60℃, and retained a 2-fold higher residual activity than the free laccase. The shift in optimum temperature of the immobilized laccase was probably due to strong attachment to the support, leading to conformational changes in the enzyme [14, 23].

After immobilization, the kinetic parameters Km and Vmax can vary considerably, depending on the type of enzyme, support materials, and process conditions. Compared with free enzyme, the Km of the immobilized laccase increased from 29.3 μM to 46.5 μM, and the Vmax was slightly lower. The lower Vmax may have resulted from mass transfer limitations and reduction in enzyme-substrate affinity after immobilization. Immobilized laccase has been reported to have up to a 13-fold higher Km on various nanoparticles [14, 22, 26], and a 92-fold higher Km was reported after immobilization on Amberlite [21]. In this study, the laccase Km increased by only 1.5-fold upon immobilization on SiO2 nanoparticles. Reductions in Vmax after laccase immobilization have been also reported, including a decrease of up to 20% [21]. The relatively low decrease in Vmax from 1,890 μmol/min/mg to 1,630 μmol/min/mg protein in this study suggests that the interaction between laccase and SiO2 nanoparticles better preserves enzyme-substrate affinity and substrate access to the active site compared with immobilization on other nanoparticles or Amberlite [21]. The thermal denaturation constant, kd, of immobilized laccase was much lower than that of the free form. This suggests that the immobilized enzyme is more stable and retains higher residual activity over a long period at temperatures in the 30-60℃ range. The t1/2 of the immobilized enzyme was up to 18-fold higher than that of the free laccase, suggesting that our immobilized system is very stable. Finally, the reusability of the immobilized enzyme has a great influence on process economics. We showed that laccase immobilized on SiO2 nanoparticles retained 94% of its initial activity after 5 cycles of use, and retained 83% activity after 10 cycles. This is much better than previous reports showing 70% and 50% residual activity under similar conditions [14, 16]. Significant losses were observed with Amberlite, where immobilized laccase retained only 30% activity after 3 cycles [21].

In conclusion, although there are many reports on the immobilization of laccase, few studies have demonstrated high efficiency along with reusability of the enzyme immobilized on SiO2 nanoparticles. The immobilized laccase system developed in this study has shown 92.9% of efficiency after immobilization and has much higher stability and reusability than free laccase, as well as laccase tested in many previous reports using other support materials such as Amberlite, magnetic-chitosan, and SiO2 composite nanoparticles. This system therefore shows promise for the development of industrial applications with high economic value.