Immobilization of laccase on functionalized multiwalled carbon nanotube membranes and application for dye decolorization

Abstract

Myceliophthora thermophila laccase was covalently immobilized on functionalized multiwalled carbon nanotubes (MWNT) arranged over a supporting membrane to obtain a permeable bio-barrier that could be applied in multibatch or continuous processes. Initially, several supporting materials, namely cellulose nitrate, agarose and polyvinyl alcohol, were evaluated as supports for the MWNT. The maximum enzyme loading of 0.286 U mg⁻¹ of MWNT was obtained for cellulose nitrate support supplemented with 0.21 mg MWNT. Immobilized laccase showed good operational stability (above 95% of its initial activity was maintained after 10 cycles of reaction) and exhibited improved stability towards temperature, pH and acetone: for instance, 21% and 49% for free and immobilized laccase, respectively, when incubated in the presence of 20% of acetone (v/v) for 6 h. The immobilized laccase was applied for Reactive Black 5 (RB5) decolorization. Maximum values of RB5 decolorization were observed in the presence of 1-hydroxybenzotriazole as mediator and at pH 5.0, with 68.09 and 84.26% decolorization after 6 and 24 h, respectively.

---

1. Introduction

Laccase (EC1.10.3.2) is a blue oxidase capable of oxidizing phenols and aromatic amines by reducing molecular oxygen to water by a multicopper system. However, some substrates with a high redox potential cannot be oxidized directly by laccase. In these cases, the using of laccase mediator systems can be an efficient solution, where mediator compounds such as hydroxybenzotriazole (HBT) act as a redox shuttle able to oxidize the target molecule. Recently, much attention has been focused on the application of laccase in different fields due to its broad substrate range, and the conversion of many substrates to unstable free radicals that may undergo further non enzymatic reactions.¹ Laccases are found to be used in dye decolorization, bioremediation, the pulp and paper industry, food processing industry, deodorants, toothpastes, mouthwashes and detergents.²

It is well known that enzymatic immobilization on a suitable support is a critical step for the efficient usage of enzymes and lots of efforts were exerted in this field. Enzyme immobilization allows efficient activity recovery, enzyme stability enhancement under harsh conditions and continuous use for multiple applications.³ The challenges of using immobilized enzymes are: to identify new matrix materials with appropriate structural characteristics, to select an immobilization strategy that improves the enzyme properties and, in addition, to understand enzyme–matrix interactions for improving the catalytic efficiency.⁴ Among the available methods, covalent binding has been the most widely used method for laccase immobilization. It allows the stabilization of the tertiary structure of the enzyme and a permanent binding to the support.

Recently, nanostructured materials have been utilized as immobilization matrices for enzymes. Among the nano-structured materials, carbon nanomaterials are the most promising candidates for enzyme immobilization because of their chemical inertness, biocompatibility, and electrical conductivity.⁵ Multiwalled carbon nanotubes (MWNT) are very promising agents because of their excellent properties, which include a unique tubular geometrical structure, superb electrical conductivity, remarkable tensile strength, and high thermal conductivity, among others. Actually, recent studies have shown that MWNT can effectively enhance direct electron transfers between electrodes and proteins.⁶

The properties of a nanomaterial, such as its surface chemistry, morphology and size, can influence the adsorption, conformation, and activity of immobilized enzymes. Moreover, nanomaterials may be easily functionalized and, therefore, their properties could also be altered.⁷ The behavior of enzymes, when their immobilization is intended, is not easy to predict and sometimes unexpected denaturation could be observed.⁸ Furthermore, the aggregation of nanoparticles may change their exposure surface, porosity, and stability, leading to altered diffusional paths and substrate accessibility to the immobilized enzyme.⁹ Regarding laccases, they have been successfully immobilized onto different nanomaterials, but mass transfer resistance was commonly observed.⁴ Therefore, more insightful studies are needed to investigate the interactions between enzymes and nanomaterials.

Many different nano materials have been studied so far. Some of the most recent and interesting ones are the so-called nanoflowers and also the magnetic nanoparticles. The former are flower-like hybrid nano morphologies with a high surface-to-volume ratio,¹⁰ whilst the latter are those nanoparticles containing magnetic elements which could allow an easy recovery of the immobilized enzyme by means of a magnetic field.^11,12 Whatever the nanomaterial employed for immobilization, its size, shape, protein ratio and other characteristics, determine its features and therefore its potential applications.

Dyes occurrence in wastewater streams generate a dangerous environmental impact ranged from photosynthesis inhibition, decreased dissolved oxygen concentration, alteration of some populations at different trophic levels, to aquatic ecosystem persistence.¹³ As a model pollutant, Reactive Black 5 (RB5), which is a common azo-dye used frequently in textile industry for dyeing processes, was used in this study in order to evaluate the efficiency of the developed laccase MWNT membrane in bioremediation treatments, since RB5 is well known as a resistant dye to biodegradation.¹⁴

In this work, we have introduced MWNT into three different membrane supports, namely cellulose nitrate, agarose, and polyvinyl alcohol (PVA), in order to immobilize laccase onto MWNT with the aim of obtaining a high enzyme loading on the carrier. The developed membranes could offer the use of immobilized laccase as a permeable bio-barrier for different redox based applications, which may allow continuous operations without requirement of subsequent complicated and time consuming separation techniques. Therefore, the aim of this work is to develop a new permeable bio-barrier (MWNT–laccase membrane), preventing the characteristic agglomeration of MWNT in solution, but with high stability and able to operate in continuous or multibatch processes.

2. Materials & methods

2.1. Chemicals

Laccase (Novozym 51003) from Myceliophthora thermophila was kindly provided by Novozymes (Denmark). 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), ethylenediamine (EDA), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma (Spain). Cellulose nitrate membranes (0.45 μm) were obtained from Sartorius AG (Germany). Bradford Protein Reagent was obtained from Bio-Rad Corporation. The other chemicals were of analytical grade.

2.2. Laccase clarification and amination

50 ml of diluted commercial laccase solution (1 : 10 with 5 mM phosphate buffer pH 7) were clarified by adsorption onto 1 g of DEAE Sepharose 6B support (GE Healthcare), at 25 °C and stirring (100 rpm) for 1 h. Then, the support was filtered and washed with the same buffer. Finally, desorption was carried out with 250 mM NaCl in 25 mM phosphate buffer, pH 7.

50 mL of clarified enzyme solution was mixed with 50 mL of a solution of 1 M EDA and 50 mM EDC at pH 4.75 for 1.5 h, and then the protein was dialyzed using dialysis cellulose membrane with molecular weight cutoff of 14 kDa (Sigma-Aldrich) in distilled water for 24 h.

2.3. Functionalization of MWNT

MWNT (15 ± 5 nm in diameter, and 5–10 μm in length), synthesized using plasma enhanced chemical vapor deposition (PECVD) were provided by Nanolab (Boston) as a powder. Carbon nanotubes were oxidized by means of the following procedure: 200 mg of MWNT were washed successively with acetone and ethanol, lyophilized (freeze-drying process) and finally were sonicated for different times (2, 4 and 6 h) in 200 mL of a mixture of H₂SO₄/HNO₃ (3 : 1); then the sample was washed first with a dilute NaOH aqueous solution and then three times with water by three centrifugation/re-dispersion cycles. Finally, the MWNT were dispersed in water, obtaining a stable dispersion of oxidized MWNT. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system with a resistivity higher than 18.2 MΩ cm.

2.4. Preparation of MWNT-membranes

Four, five and six milliliters of a previously prepared dispersion containing 0.053 mg mL⁻¹ of MWNT were injected to a cellulose nitrate membrane (Sartorius, 25 mm diameter, 0.45 μm pore size) to reach final concentrations of 0.21, 0.26 and 0.32 mg of MWNT/membrane. Then the MWNT-membrane was left to dry at room temperature for at least 24 h prior their use.

MWNT/agarose membranes were obtained by means of the following protocol; 150 mg of agarose were dissolved in 15 mL of Milli-Q water, then the solution was heated at 50 °C and stirred for 30 min. Then 1 mL of MWNT dispersion (0.8 mg mL⁻¹) was added to the previously prepared agarose solution. The mixture was cooled at room temperature and thenfrozen at −20 °C for 18 h. Finally, the water was removed by lyophilization for 24 h. The same procedure was used for MWNT/polyvinyl alcohol (PVA) membranes but using 750 mg of PVA instead of 150 mg of agarose.

2.5. Laccase immobilization on MWNT membranes

The diluted aminated laccase and EDC (50 mM) were mixed in 100 mM phosphate buffer (pH 5.0) and forced to pass through the membranes for 3 h at room temperature (25 °C) with a flow rate of 30 ml h⁻¹. After immobilization, the membranes were washed with 50 mM phosphate buffer (pH 7.0), then with 100 mM citrate buffer (pH 4.5) and finally with 50 mM phosphate buffer (pH 7.0). All experiments were performed in triplicate.

2.6. Laccase assay and protein estimation

The free laccase activity was assayed spectrophotometrically (Jasco, V-630 UV-Vis Spectrophotometer, Japan) with ABTS (0.58 mM) as substrate in 100 mM citrate buffer at pH 4.5 and 25 °C. The change in absorbance at 436 nm (ε = 29 300 M⁻¹ cm⁻¹) was monitored for 1 min. One unit (U) was defined as the amount of enzyme that oxidized 1 μmol of ABTS per min. To measure immobilized laccase activity, the support was mixed with 10 mL of 0.58 mM ABTS in 100 mM citrate buffer (pH 4.5) at 25 °C, under orbital stirring at 50 rpm. Samples were taken every minute and absorbance was measured at 436 nm.

Protein concentration was determined by the method described by Bradford¹⁵ using Bio-Rad Protein Assay Reagent and BSA as standard protein. The quantity of protein bound to the support was calculated by subtracting the protein recovered in the combined washing of the support–enzyme complex from the total protein content used for immobilization.

2.7. Operational stability of immobilized laccase

The MWNT membranes were immersed into fresh substrate solution (10 mL of 0.58 mM ABTS in 100 mM citrate buffer, pH 4.5) for 10 cycles (5 min each) at 25 °C, under orbital stirring at 50 rpm. During each cycle, samples were withdrawn each minute for absorbance measurement and then returned into the reaction mixture. At the end of each cycle, the reaction was stopped by substrate removal. Then, the immobilized enzyme was washed 4 times with distilled water to start a new cycle. All experiments were performed in triplicate.

2.8. Effect of temperature and thermal stability

Temperature effect on activity was studied at 40–90 °C at pH 7.0. Thermal stability was determined by incubation in 50 mM phosphate buffer (pH 7.0) at 50, 60, 70 and 80 °C; samples were taken at different intervals during 48 h for laccase activity determination.

Thermal inactivation parameters were determined according to Ladero et al.¹⁶ by Excel Solver software for nonlinear regression.

2.9. pH activity profile and stability

Activity at pH values (pH 2.0–7.0) were recorded using Robinson buffer. The pH inactivation was determined by incubation of free and immobilized laccase in 100 mM Robinson buffer (pH 3.0, 5.0, 7.0, and 9.0) at 50 °C for different time intervals up to 120 h. All experiments were performed in triplicate.

2.10. Stability of laccase in the presence of acetone

Free and immobilized laccase were incubated in 50 mM phosphate buffer pH 7.0, at 55 °C, for 48 h, with different acetone concentrations (10, 20, and 50% (v/v)). Residual activities were determined at regular time intervals. All activity measurements were performed in triplicate.

2.11. RB5 decolorization

Decolorization of RB5 (700 mg L⁻¹) was carried out in presence of HBT (1–5 mM) at different buffered pH values (pH 3.0, 5.0, 7.0, and 9.0), 25 °C or 50 °C and shaking (100 rpm) up to 24 h of reaction. Decolorization was initiated by free aminated laccase addition (0.15 or 1 U). For immobilized enzyme, 0.9 U of laccase immobilized on membranes with 0.21 mg MWNT were used instead. Control experiments, without enzyme, were carried out in parallel.

Decolorization was measured as the reduction of absorbance, in percentage, of the reaction mixture at the maximum absorption wavelength of the dye.

Decolorization with immobilized laccase was followed at periodic intervals for each cycle of 2 h. After each cycle, the reaction mixture was discarded and immobilized laccase was reused for another cycle of decolorization under the same conditions. The process was repeated up to 4 cycles to test the reusability of immobilized laccase in RB5 decolorization. Identical experiments were performed in parallel by using membranes without immobilized enzyme.

3. Results & discussion

3.1. Immobilization of laccase

Single walled carbon nanotubes tend to form large agglomerates in solution because of intermolecular interactions, which may result in a lower access of immobilized laccase to the substrate.⁹ To avoid this problem, we propose the use of permeable membranes with laccase immobilized onto MWNT. For that purpose, different membranes namely cellulose nitrate, agarose and PVA were evaluated as MWNT–laccase support. Both agarose and PVA membranes have a similar structure, with big pores and nanotubes embedded into the membrane (Fig. 1a, b and d). Thus, the nanotube surfaces are not fully available for laccase attachment. On the contrary, the bare carbon nanotube membranes supported in cellulose nitrate have a completely different structure (Fig. 1c, e and f) with the full nanotube surface available for laccase immobilization.

Fig. 1 Digital images of pure agarose (a), MWNT/agarose (b), MWNT/cellulose (c) membranes, and scanning electron microscopy (SEM) images of MWNT/agarose (d) and MWNT/cellulose (e and f).

The chemical inertness of carbon nanotubes requires preliminary chemical modifications to increase their reactivity. Thus, MWNT were carboxylated since carboxylic groups provide ideal binding points for the covalent immobilization of proteins using the cross-linker EDC. Laccase coupling onto MWNT was achieved in a single step, where this enzyme and EDC were mixed in a single vessel and passed through MWNT membranes. The previous amination of laccase is expected to improve the amount of immobilized laccase by enhancing the interaction of the oxidized nanotubes with the amino groups of the enzyme.

Laccase covalent binding results onto the three different membranes are summarized in Table 1. A narrow range of MWNT amount (0.21–0.32 mg) was tested due to experimental limitations, namely physical retardation during membrane preparation or non-homogenous distribution of MWNT, in case of higher and lower MWNT amounts, respectively. The maximum enzyme loading (0.286 U mg⁻¹ MWNT) was obtained for cellulose nitrate membrane with 0.21 mg MWNT. Therefore these conditions were selected for further studies. Both agarose and PVA partially coat the nanotube surface limiting their interaction with enzymes, so their corresponding membranes were then discarded for additional experiments.

Table 1 M. thermophila laccase activity on different immobilization supports

Support	Total laccase activity (U)	Laccase activity (U mg^−1 MWNT)
Cellulose nitrate membrane with 0.21 mg MWNT	0.060	0.286
Cellulose nitrate membrane with 0.26 mg MWNT	0.069	0.265
Cellulose nitrate membrane with 0.32 mg MWNT	0.079	0.247
Agarose disc with 0.21 mg MWNT	0.024	0.114
PVA disc with 0.21 mg MWNT	0.019	0.090

---

3.2. Operational stability of immobilized laccase

The stability of the immobilized enzyme after repeated use provides information about the resistance of catalyst activity and also about reusability effectiveness. As can be seen from Table 2, different operational stability was obtained depending on the tested support; these differences could be related to the specific nature of each support. Although a significant decrease in operational stability was observed for cellulose nitrate membranes with high amounts of MWNT (0.26, 0.32 mg), immobilized laccase maintained above 95% of its initial activity after ten cycles in the case of cellulose nitrate membrane with 0.21 mg MWNT.

Table 2 Operational stability of laccase immobilized on various carbon nanomaterials

Cycle	U mg^−1 MWNT
	MWNT cellulose nitrate			MWNT PVA	MWNT agarose
	0.21 mg	0.26 mg	0.32 mg
1	0.286 ± 0.13	0.265 ± 0.08	0.247 ± 0.11	0.092 ± 0.01	0.143 ± 0.11
2	0.262 ± 0.11	0.219 ± 0.05	0.195 ± 0.02	0.113 ± 0.02	0.100 ± 0.14
3	0.290 ± 0.12	0.212 ± 0.06	0.163 ± 0.01	0.127 ± 0.01	0.100 ± 0.09
4	0.224 ± 0.07	0.215 ± 0.06	0.205 ± 0.04	0.133 ± 0.02	0.095 ± 0.05
5	0.233 ± 0.06	0.212 ± 0.07	0.200 ± 0.03	0.141 ± 0.00	0.081 ± 0.12
6	0.257 ± 0.07	0.200 ± 0.05	0.163 ± 0.01	0.146 ± 0.01	0.052 ± 0.06
7	0.281 ± 0.07	0.196 ± 0.06	0.172 ± 0.04	0.145 ± 0.02	0.052 ± 0.08
8	0.295 ± 0.05	0.188 ± 0.06	0.173 ± 0.03	0.148 ± 0.00	0.043 ± 0.06
9	0.276 ± 0.07	0.173 ± 0.06	0.169 ± 0.04	0.159 ± 0.02	0.033 ± 0.07
10	0.281 ± 0.06	0.154 ± 0.04	0.138 ± 0.08	0.148 ± 0.00	0.005 ± 0.02

---

An unexpected behavior of the PVA membrane was observed since it increased the enzymatic activity as the recycling cycles go further. This effect could be ascribed to a possible modification of mass transfer capability or to the reorientation of the enzyme. PVA has not behave as an inert support in this case, and it even may act as an inducer for laccase activity, as it was previously stated.¹⁷

Therefore, our results show that the reusability of the immobilized laccase can be remarkably extended in the presence of the proper MWNT amount. The improvement of laccase operational stability by immobilization in MWNT was also tested, for instance, by Xu et al.,¹⁸ who obtained a 72.7% of initial activity after 7 cycles. Similarly, the immobilized laccase on Eupergit C support retained 65% of initial activity after 10 cycles.¹⁹

3.3. Effect of temperature on laccase activity and stability

No remarkable differences on the laccase activity values were observed for immobilized and free laccases (Fig. 2). M. thermophila laccase activity progressively increased with the rise of reaction temperature up to 70 °C, which is the optimum value. The denaturation makes the activity to drop down at higher temperatures.

Fig. 2 Effect of temperature on laccase activity.

Regarding thermal stability, M. thermophila laccase retained its activity after heat exposure to 50 and 60 °C for 4 h by about 88.13 and 65.02% for free enzyme and 90.98 and 78.95% for immobilized enzyme, respectively (Fig. 3).

Fig. 3 Thermal stability of M. thermophila laccase (A) free, (B) immobilized.

For longer times, more specifically after 48 h of incubation and at 50 and 60 °C, free laccase retained only 26.66 and 1.86% of its activity, whereas immobilized laccase on MWNT (0.21 mg) cellulose nitrate membranes retained 72.93 and 23.12%. Therefore, the residual activity of immobilized laccase was approximately 2.7 and 12.4-fold higher than that of the free enzyme. These results not only show a positive effect of immobilization, but also that the enzyme under investigation is more stable than other similar enzymes. For instance, T. versicolor laccase have shown residual enzymatic activities of 37.8 and 32.2% for the free laccase and 78.5 and 52.5% for the immobilized enzyme on SiO₂ nanoparticles at 50 and 60 °C, respectively.²⁰

Thermal inactivation for free and immobilized enzymes was properly fitted to first-order model eqn (1) and (2), which implies a one-step transition between the active and denatured state (Table 3).

a = (1 − α)e^−k₁t + α (1)

where a is the residual activity percentage of initial activity, α is the remaining activity after the partial enzyme inactivation, k₁ is the first-order rate coefficient, and t is the time.

Table 3 Parameters of first-order model for thermal inactivation¹⁶ of free and immobilized laccase: remaining activity after the partial enzyme inactivation (α₁), inactivation rate constant (k₁), and activation energy (E_a)^a

Temperature (°C)	Free laccase				Immobilized laccase
	α1	k1 (h^−1)	R^2	Ea (kJ mol^−1)	α1	k1 (h^−1)	R^2	Ea (kJ mol^−1)
a Each value represents the mean of triplicate measurements and varies from the mean by not more than 10%.
50	0.0477	0.0279	0.995	135.73	0.7245	0.0707	0.984	133.91
60	0.0102	0.1048	0.994		0.2441	0.0733	0.968
70	0.0086	0.5677	0.995		0.1283	0.3137	0.987
80	0.004	1.8703	0.9999		0.0433	1.1318	0.983

---

Arrhenius equation

k₁ = k₀e^−E_a/RT (2)

where k₀ is the pre-exponential factor, E_a is the energy of activation, T is the temperature (K) and R is the universal gas constant (8.314 J mol⁻¹ K⁻¹).

The kinetic data show that k values increase in a higher extent for the free enzyme than for the immobilized one as temperature rises, which demonstrates the enhancement of laccase thermal stability by immobilization.

3.4. Effect of pH value on laccase activity and stability

The pH optimum for laccases depends on the substrate and on its redox potential. Fungal laccases are usually stable at acidic pH, although pH stability varies considerably depending on the source of the enzyme.²¹ In the present study, the optimum activity was recorded at pH 3.0 for both free and immobilized laccase, with a higher activity level in case of the immobilized enzyme at acidic region, which may be related to its stability (Fig. 4).

Fig. 4 Effect of pH value on the activity of free and immobilized M. thermophila laccase using ABTS as substrate.

Patrick et al.²² stated that the pH profile is the result of two opposing effects: the first effect is due to the redox potential difference between a reducing substrate (phenolic compound) and the Type 1 copper center of laccase, where the electron transfer rate is favored for phenolic substrates at a high pH; the second effect is generated by the binding of a hydroxyl anion to the type 2/Type 3 copper centers of laccase, which inhibits the binding of O₂, the terminal electron acceptor. Therefore the activity at alkaline pH could be inhibited because of the increased amount of hydroxyl ions.²³ However, from the stability data obtained, we can state that the M. thermophila laccase is stable at alkaline range. For instance, the free laccase retains about 62.4, 85.6, and 90.3% of the initial activity after 3 h of incubation at pH 5.0, 7.0, and 9.0. Furthermore, the immobilized enzyme was more stable by 1.27, 1.14, and 1.06 times than the free enzyme, under the same conditions. After 48 h of incubation, the free enzyme retained 29.33, and 58.85% of initial activity at pH 7.0 and 9.0 whereas, the immobilized laccase retained 77.96, and 80.90% respectively, which reflect the success of immobilization process in enhancement of enzyme stability (Fig. 5).

Fig. 5 pH stability of M. thermophila laccase (A) free, (B) immobilized.

3.5. Stability of free and immobilized laccase in acetone

The use of laccase in organic solvents extend its potential applications and could have various advantages such as elimination of microbial contamination, increase the solubility of nonpolar substrates, repression of water-dependent side reactions, and catalysis of non aqueous chemical reactions.²⁴

In this context, we have studied the ability of M. thermophila laccase to retain its activity in aqueous acetone. When acetone concentration was increased, the activity of laccase decreased dramatically (Fig. 6).

Fig. 6 Stability of M. thermophila laccase against acetone (A) free, (B) immobilized.

The results obtained indicated that the immobilized laccase was highly stable at the lowest concentration of acetone tested (10% v/v) and it is, in general terms, more stable than the free enzyme. For instance, the half-life times of the immobilized laccase were 26.36, 5.91, and 1.26 h at 10, 20, and 50% (v/v) acetone respectively, whereas in the case of the free laccase they were 5.41, 1.67, and 1.04 h at the same conditions. Therefore, laccase stability in water–acetone mixtures was improved by immobilization on MWNT cellulose nitrate membrane. Although previous reports²⁵ stated that 50% (v/v) acetone usually inhibit the activity of oxidases, probably by destructing the molecular interactions which determine the protein structure, we were able to improve the stability at these harsh conditions.

3.6. RB5 decolorization

As a previous study of RB5 decolorization with immobilized laccase, we have assessed the effect of pH (3.0–9.0) with free laccase. Maximum decolorization was observed at pH 5 and it was, therefore, selected for further experiments. Regarding immobilized laccase, temperature (25 and 50 °C), laccase activity (0.9 U) and HBT (0, 1 and 5 mM), were selected as main factors in order to study RB5 decolorization.

The presence of HBT was necessary in order to decolorize RB5 with immobilized laccase from M. thermophila, since no decolorization was detected, even after 20 h of reaction, without mediator addition. This effect has been also observed by Kunamneni et al.²⁶ 5 mM HBT allows getting the best decolorization results; however 1 mM HBT allows to get 0.95-fold the maximum decolorization in the conditions tested. In addition, 1 U of laccase and 50 °C were the selected values in order to improve the decolorization value. The best decolorization values rised around 60 and 75% after 2 and 24 h of reaction respectively. From the whole results (data not shown), temperature and enzyme activity were the main factors affecting RB5 decolorization with immobilized enzyme.

3.7. Reusability of immobilized laccase in RB5 decolorization

The advantage of immobilized laccase is not only the stability increase, but also its reusability. Hence, the decolorization of RB5 by the immobilized laccase on MWNT membranes was studied through successive cycles. After the first cycle of decolorization (2 h), the percentage of RB5 decolorization was 62.73% which revealed that immobilized laccase has potential application in dyestuff treatment (Fig. 7).

Fig. 7 Reusability of immobilized laccase in RB5 decolorization.

By the end of the 2^nd cycle of using the immobilized laccase, the decolorization percentage was significantly reduced (33.18%) and even more in the next cycles (6.89% after the 4^th cycle). Therefore, although the possibility of reusing the immobilized laccase in RB5 decolorization is a fact, a decrease in effectiveness was observed. The inactivation of laccase by the selected mediator has been reported for P. cinnabarinus laccase.²⁷ However, this possible effect does not seem to explain the high drop in decolorization capability. Moreover it is known that anionic sulphonic dyes, RB5 for instance, may protect enzymes from inactivation, since they can form ion pairs with positively charged protein groups, improving the enzymatic stability.²⁸ Recently, some studies have shown similar limitations in the reusability of immobilized laccase that could explain the effect presented in this paper. Carbamazepine was degraded by this methodology with a significant decrease in efficiency after each cycle,²⁹ which was attributed to the high molar mass products formed during carbamazepine oxidation, which could bury the enzyme and limit the mass transfer. A similar effect was also explained for PAH oxidation,³⁰ since an active site poisoning produced by deposition of any PAH oxidation intermediate was proposed as the reason for the limited reusability. Therefore, the reaction products could act as laccase inhibitor, which is an important limitation for the application of immobilized laccase for environmental purposes.

4. Conclusions

Laccase can be covalently immobilized using MWNT in three different like-membrane materials. Two of them, MWNT/agarose and MWNT/PVA, showed a significant drawback since both agarose and PVA partially covers the nanotube surface, limiting the anchoring of enzymes on the membrane. On the contrary, the developed MWNT/cellulose–nitrate membranes are characterized by reusability, remarkable pH, thermal, and chemical stability in joint with a superior loading capacity of MWNT. These membranes may work as promising permeable bio-barrier, tackling some industrial challenges regarding biotechnological applications of immobilized enzymes. As a model pollutant, RB5 was effectively decolorized by laccase immobilized onto MWNT/cellulose–nitrate membranes, although this process lacks of operational stability, which indicates that reusability of laccase immobilized onto MWNT/cellulose–nitrate membranes depends on the substrate to be oxidized. New improvements in either immobilization process or operational conditions are necessary in order to get the desired reusability for the repeated degradation of RB5 and similar compounds.

Acknowledgements

Abdelmageed M. Othman is grateful to the Partnership and Ownership Initiative (ParOwn) program (Egypt), for his fellowship. This work was partially funded by the project EM2014/041 (Xunta de Galicia, Spain).

References

1. E. P. Cipolatti, M. J. A. Silva, M. Klein, V. Feddern, M. M. C. Feltes, J. V. Oliveira, J. L. Ninow and D. Oliveira, J. Mol. Catal. B: Enzym., 2014, 99, 56.
2. M. Fernández-Fernández, M. A. Sanromán and D. Moldes, Biotechnol. Adv., 2013, 31, 1808.
3. F. D'acunzo, C. Galli and B. Masci, Eur. J. Biochem., 2002, 269, 5330.
4. R. Shekher, S. Sehgal, M. Kamthania and A. Kumar, Enzyme Res., 2011, 217861.
5. R. V. Mundra, X. Wu, J. Sauer, J. S. Dordick and R. S. Kane, Curr. Opin. Biotechnol., 2014, 28, 25.
6. R. J. Lopez, S. Babanova, Y. Ulyanova, S. Singhal and P. Atanassov, ChemElectroChem, 2014, 1, 241.
7. S. Boncel, A. Zniszczoł, K. Szymańska, J. Mrowiec-Białoń, A. Jarzębski and K. Z. Walczak, Enzyme Microb. Technol., 2013, 53, 263.
8. C. Zhang, S. Luo and W. Chen, Talanta, 2013, 113, 142.
9. R. Pang, M. Li and C. Zhang, Talanta, 2015, 131, 38.
10. S. W. Lee, S. A. Cheon, M. I. Kim and T. J. Park, J. Nanobiotechnol., 2015, 13, 54.
11. E. González-Domínguez, M. Comesaña-Hermo, R. Mariño-Fernández, B. Rodríguez-González, R. Arenal, V. Salgueiriño, D. Moldes, A. M. Othman, M. Pérez-Lorenzo and M. A. Correa-Duarte, ChemCatChem, 2016, 8, 1264.
12. V. V. Kumar and H. Cabana, Bioresour. Technol., 2016, 200, 81.
13. R. A. Damodar and S. J. You, Sep. Purif. Technol., 2010, 71, 44.
14. K. Murugesan, A. Dhamija, I. H. Nam, Y. M. Kim and Y. S. Chang, Dyes Pigm., 2007, 75, 176.
15. M. M. Bradford, Anal. Biochem., 1976, 72, 248.
16. M. Ladero, G. Ruiz, B. C. C. Pessela, A. Vian, A. Santos and F. Garcia-Ochoa, Biochem. Eng. J., 2006, 31, 14.
17. N. A. Mohidem and H. B. Mat, J. Sol-Gel Sci. Technol., 2012, 61, 96.
18. R. Xu, R. Tang, Q. Zhou, F. Li and B. Zhang, Chem. Eng. J., 2015, 262, 88.
19. L. Lloret, F. Hollmann, G. Eibes, G. Feijoo, M. T. Moreira and J. M. Lema, Biodegradation, 2012, 23, 373.
20. S. K. S. Patel, V. C. Kalia, J. H. Choi, J. R. Haw, I. W. Kim and J. K. Lee, J. Microbiol. Biotechnol., 2014, 24, 639.
21. P. Baldrian, FEMS Microbiol. Rev., 2006, 30, 215.
22. F. Patrick, G. Mtui, A. M. Mshandete, G. Johansson and A. Kivaisi, Afr. J. Biochem. Res., 2009, 3, 250.
23. C. Munoz, F. Guillén, A. T. Martínez and M. J. Martínez, Appl. Environ. Microbiol., 1997, 63, 2166.
24. N. Doukyu and D. Ogino, Biochem. Eng. J., 2010, 48, 270.
25. I. F. Fiţigău, F. Peter and C. G. Boeriu, Acta Biochim. Pol., 2013, 60, 817.
26. A. Kunamneni, I. Ghazi, S. Camarero, A. Ballesteros, F. J. Plou and M. Alcalde, Process Biochem., 2008, 43, 169.
27. D. Ibarra, J. Romero, M. J. Martínez, A. T. Martínez and S. Camarero, Enzyme Microb. Technol., 2006, 39, 1319.
28. D. Matulis, C. G. Baumann, V. A. Bloomfield and R. E. Lovrien, Biopolymers, 1999, 49, 451.
29. C. Ji, J. Hou, K. Wang, Y. Zhang and V. Chen, J. Membr. Sci., 2016, 502, 11.
30. L. F. Bautista, G. Morales and R. Sanz, Chemosphere, 2016, 136, 273.

---