Adsorption of Laccase on Multi-walled Carbon Nanotubes

Abstract

Controlling redox enzyme adsorption on an electrode surface is a key feature for designing efficient and stable bioelectrodes for biofuel cell applications. Here, we report an analysis of the adsorption mechanism of laccase, which catalyzes the 4-electron reduction reaction of oxygen, on multi-walled carbon nanotubes (MWCNTs). The adsorption of laccase on the surface of MWCNTs can be explained by the Langmuir model with a pseudo-first-order kinetics. We found that laccase adsorption is an exothermic process, mainly driven by hydrophobic interactions between laccase and the MWCNTs. Our investigation also revealed that electrostatic interactions are not the main driving forces and play a small role in laccase adsorption. A clear understanding and devising an efficient method of enzyme adsorption will provide important guidelines for optimizing, not only the surface material design but also the adsorption method for high-performance electrodes.

1. Introduction

Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2, a blue multi-copper oxidase) is an enzyme found in various plants, fungi, and bacteria.^1,2 This enzyme has been extensively studied as a potential biocatalyst for the molecular oxygen reduction reaction at the cathode in enzymatic biofuel cell (EBFC) applications.^3,4 Some EBFCs have been designed as power sources for implanted medical devices by generating electric power from the oxidation of abundantly bioavailable glucose and the reduction of molecular oxygen in living organisms.⁵ However, development and applications of EBFCs have encountered some critical challenges, mainly arising from low output current and short lifetimes of the enzymes used.⁶ The current values depend on the amount of enzyme, as well as the orientation and stability on electrode surfaces.⁷ In general, enzyme immobilization on electrode surfaces represents the most promising strategy to preserve the conformational structure of the enzymes and thus, enhance the lifetime of enzymatic electrodes.⁸ Although there are many strategies for enzyme immobilization, the most popular and efficient technique is physical adsorption, which is a simple method based on the interactions between the enzymes and the carrier surface and does not require enzyme pre-treatments or chemical modifications.⁹ In the case of laccases, the adsorption of laccase on electrode surfaces, such as carbon nanotubes (CNTs), has been intensively investigated to design biocathodes for EBFC applications and biosensors.¹⁰ However, few reports have addressed the mechanism and kinetics of laccase adsorption onto CNTs. In most cases, the existing methods and protocols for laccase adsorption on CNTs (and other materials) are almost identical and do not consider the specificity of each enzyme or the properties of the CNTs.

Enzyme adsorption onto CNTs can be evaluated as a function of the number of desorbed (or unmodified) enzymes under operational conditions. From a thermodynamic perspective, the physical adsorption of enzymes on a solid surface generally reaches an adsorption/desorption equilibrium, when both adsorption and desorption of the enzymes on the CNTs occur.^10–12 Enzyme adsorption on solid surfaces may depend on various factors, including affinity with the carrier surface, enzyme concentration, pH, and temperature of the system.¹³ Optimization of laccase adsorption and minimization of desorption are the main issues in the design of high-performance laccase-based biocathodes. Silva et al. studied the effect of multi-walled carbon nanotube (MWCNT) surface chemistry on the efficiency of non-covalent laccase immobilization and the catalytic activity of the resulting hybrid complexes.¹⁴ Strong π–π stacking and hydrophobic interactions between the enzyme and pristine MWCNTs were observed; these characteristics were related to the specific properties of both the MWCNTs and laccase. In a previous study on laccase adsorption on MWCNTs, we demonstrated that the most efficient laccase immobilization was obtained using functionalized MWCNTs; however, in terms of long-term stability, pristine MWCNTs exhibited better laccase stability on the electrode.¹⁵ The improved stability was explained by hydrophobic interactions between laccase and pristine MWCNTs, rather than ionic interactions; however, in the case of functionalized MWCNTs, laccase adsorption/desorption is highly sensitive to the ionic strength of the buffer solution. In this study, we investigated the kinetics and thermodynamics of laccase adsorption on MWCNTs under different conditions, including initial laccase concentration, pH, temperature, and ionic strength. The clear understanding and analytical method of enzyme adsorption will provide important knowledge for optimizing not only the carrier support design but also the adsorption method towards devising high-performance enzyme electrodes.

2. Materials and Methods

2.1 Chemicals

Laccase (from Trametes sp.) was kindly provided by Amano Enzyme, Inc. (Nagoya, Japan). 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), NaCl, Na₂HPO₄ (>99 %), NaH₂PO₄ (>99 %), and acetic acid (≥99.8 %) were purchased from Sigma Aldrich. All aqueous solutions were prepared using Nanopure water with a resistivity of 18.2 MΩ cm at 25 °C (Purelab Prima, Décines-Charpieu, France). Multi-walled carbon nanotubes (MWCNTs) and amine-functionalized MWCNTs (MWCNTs-NH₂) (each approximately 10–15 nm in diameter, >95 % purity, Nanocyl S.A., Sambreville, Belgium) were used without modification. 0.1 M (M = mol L⁻¹) sodium acetate buffer solution of pH 5.0 was prepared by mixing 35.2 mL of 0.1 M sodium acetate and 14.8 ml of 0.1 M acetic acid.

2.2 Purification of laccase

5.0 g of laccase powder, which contains a large number of stabilizers, was solubilized in 50 mL of 0.1 M acetate buffer, pH 5.0 in a 50 mL test tube with a conical base. Subsequently, the suspension was vortexed for 5 min at 4 °C and centrifuged for 15 min at 10,000 × g. The supernatant was then extracted and poured into two 15 mL ultrafiltration centrifuge tubes (Amicon Ultra-15 Centrifugal Filter Units). Both the tubes were then centrifuged at 6,000 rpm at 4 °C for 30 min cycles until the total volume was reduced by a factor of ten. The concentrate was then extracted and aliquoted. The concentration of the purified laccase was then measured by spectrophotometry with values of 27.9 mg mL⁻¹.

2.3 Laccase adsorption

Physical adsorption was carried out by incubating MWCNTs in laccase solution with gentle stirring for 12 h, filtering with a vacuum pump, and then drying at ambient temperature. For spectrophotometric studies, pristine MWCNTs (3.0 mg) were incubated in 2.0 mL buffer solution (pH 4.5) containing laccase at different concentrations varying from 500–10,000 mg L⁻¹. After 5 h, the MWCNTs were filtered and the supernatants were collected for analysis, with a UV-Vis spectrophotometer. To assess the effect of temperature on laccase adsorption on MWCNTs, three tubes containing 2.0 mL of acetate buffer (pH 4.5), 1.5 mg mL⁻¹ MWCNTs, and 2,000 mg L⁻¹ laccase were prepared. Each tube was incubated for 5 h at 37, 20, or 4 °C, respectively. The experiments were repeated three times.

2.4 Spectrophotometric measurements and laccase activity determination

An ultraviolet-visible (UV-Vis) spectrophotometer (Jenway, France) was used to study the specific activity of free laccase in the solution phase and determine the quantity of laccase adsorbed on the MWCNTs over time. Activity of the laccase solution without MWCNTs over time was used as a control. The specific activity of free laccase in phosphate buffer solution was determined by using ABTS as substrate for the enzymatic reduction of dioxygen into water. By monitoring the absorption at 415 nm of oxidized form of ABTS laccase activity was estimated. In the presence of WCNTs, the decrease in laccase apparent activity in the sample with MWCNTs corresponds to a decrease in its free concentration in the solution, which results from laccase adsorption on MWCNTs.

3. Results and Discussion

3.1 Laccase adsorption/desorption isotherms

As shown in Fig. 1a, the amount of adsorbed laccase at the equilibrium state (q_e, mg g⁻¹) increased as the laccase concentration (c₀) increased up to 5000 mg L⁻¹, and thereafter became constant at higher concentrations. The existence of an adsorption plateau indicates that the adsorbing surface is saturated with laccase molecules, and any further increase in laccase concentration would not affect the amount of adsorbed laccase.¹⁶

Figure 1.

Adsorption isotherms for laccase on MWCNT at different concentrations of laccase (pH 4.5, 20 °C). (a) Effect of initial laccase concentration (c₀) on the amount of adsorbed laccase on pristine MWCNTs. (b) amount of adsorbed laccase at the equilibrium state (q_e) vs. the concentration of laccase in the solution at the equilibrium state (c_e) plot and regression curves. Solid curve represents Eq. 1 and curve with broken line represents Eq. 2 in the text. The error bars were evaluated by the Student t-distribution at 90 % confidence level.

The obtained data were fitted using Langmuir and Freundlich isotherm models.¹⁷ Figure 1b shows the plot of q_e versus c_e with a non-linear regression curve using Eq. 1 in the Langmuir and Freundlich isotherm models. The Langmuir isotherm model can be expressed by the following equation:

  

\begin{equation} q_{\text{e}} = \frac{K_{\text{L}}q_{\max}c_{\text{e}}}{1 + K_{\text{L}}c_{\text{e}}} \end{equation} (1)

where q_e (mg g⁻¹) is the amount of enzyme adsorbed per unit mass of adsorbent at equilibrium, c_e (mg L⁻¹) is the concentration of laccase in the solution at the equilibrium state ($ = c_{0} - q_{\text{e}} \times \frac{\text{weight of MWCNT}}{\text{volume of the solution}}$), q_max (mg g⁻¹) is the maximum adsorption capacity, and K_L is the Langmuir constant.

The Freundlich model, which is also widely used to explain adsorption, explains the relationship between q_e and c_e as follows:

  

\begin{equation} \log(q_{\text{e}}) = \frac{1}{n}\log(c_{\text{e}}) + \log(k_{\text{F}}) \end{equation} (2)

where c_e (mg L⁻¹) is the equilibrium concentration, and n and K_F are Freundlich constants related to the intensity of the adsorption and sorption capacity, respectively.

The Langmuir model reproduced the results with the following fitting parameters: q_max = 1580 mg g⁻¹ and K_L = 0.00045 L g⁻¹. If we consider that the Brunauer–Emmett–Teller (BET) specific surface area of MWCNTs is approximately 250 m² g⁻¹; laccase has a spherical form with a diameter of 10 nm; and that the molecular weight of laccase is 60,000 Da, the adsorption capacity represented only 20 % of the surface of MWCNTs. This phenomenon can be explained by the fact that MWCNTs were not completely dispersed in the buffer solution but rather formed aggregates with a smaller amount of exposed active surface area than the actual surface area of the total number of dispersed MWCNTs. The Langmuir isotherm model corresponds to the formation of a monolayer of adsorbate on the outer surface of the adsorbent. Once a monolayer is formed on the surface of the MWCNTs, no further adsorption can occur.

3.2 Kinetics of laccase adsorption on MWCNTs

The essential parameter for any adsorption kinetics is the number of protein molecules adsorbed per unit area in any increment of time, dΓp/dt. It is generally accepted that the kinetic process of adsorption diffusion models is mainly controlled by the processes of liquid film diffusion and intra-particle diffusion; one of these processes should be the rate-limiting step.¹⁸ Kinetics of laccase adsorption was determined by measuring the amount of laccase adsorbed at 20 °C for 5 h (Fig. 2a), which allowed us to identify the mechanism of the first phase of laccase adsorption onto the MWCNTs. The results showed that the amount of adsorbed laccase increased with increasing incubation time and reached a maximum value of 515 ± 18 mg laccase/g MWCNT after 3 h. This phase was followed by slight desorption during the next hour. The adsorbed laccase reached a plateau of 507 ± 16 mg g⁻¹ at 5 h, corresponding to a saturation phase (Fig. 2a). Generally, protein adsorption kinetics are monotonically increasing curves that, after a sufficiently long time, reach saturation where the number of adsorbed proteins corresponds to the saturation level of the surface.^19,20 In this case, overshooting was observed, where the amount of adsorbed laccase reached a maximum during laccase adsorption. This phenomenon has been reported for different proteins and is attributed to the reorganization of proteins on the surface of the carrier during adsorption. Overshooting during the adsorption process has also been extensively reported in case of colloid and polymer adsorption and is mechanistically explained by the so-called time-delay model. This model suggests that adsorption begins when desorption from the surface is prohibited. After a certain time delay, desorption starts due to conformational rearrangement, which may cause overshoot if the surface is substantially covered and thus oversaturated. Previous studies with kinetics of lysozyme adsorption onto hydrophobic surfaces has shown that kinetic overshoots are observed that can be explained by an interfacial relaxation from an end-on to a side-on orientation, which occurs by rollover and not by the displacement of end-on adsorbed proteins by side-on adsorbed proteins.²¹ The same overshooting phenomena was also observed during the adsorption of lysozyme on the silica surface.²²

Figure 2.

Kinetics of Laccase Adsorption on MWCNTs. (a) Adsorption of laccase on MWCNTs at different times in a buffer (pH 4.5). The solid curve represents the regression curve based on the first-order kinetic model and the broken curve represents the regression curve based on the second-order model with best fitting parameters. (b) The intra-particle diffusion plot of q_t vs. t^1/2 revealed a two-phase process. The error bars were evaluated by the Student t-distribution at 90 % confidence level.

Vroman et al. observed that fibrinogen rapidly adsorbs to the solid surface, but after a short time, it passes through a coverage maximum and finally covers the surface in smaller amounts at the equilibrium state than at the intermediate state.²³ Rabe et al. studied the adsorption kinetics of the model protein β-lactoglobulin on a hydrophilic glass surface using fluorescence detection, and suggested a reliable explanation of the overshooting effect, that combines the idea of orientation or conformational rearrangements with some aspects of the time delay model.²⁰ After the initial irreversible phase of protein’s physical adsorption to the surface, a certain coverage level exceeds during adsorption, and the binding behavior changes from irreversible to reversible [19]. In contrast, some previous explanations suggest that, the formation of the first irreversible protein layer is followed by a second reversible layer.²⁴

The time dependence of laccase adsorption on MWCNTs was analyzed based on the Langmuir model with simple kinetic models, namely, the pseudo-first-order and pseudo-second-order models. The first-order kinetic model, q_t, can be expressed by the following equation:

  

\begin{equation} \ln\left(\frac{q_{\text{e}} - q_{\text{t}}}{q_{\text{e}}}\right) = -k_{1}t \end{equation} (3)

where k₁ (min⁻¹) is the rate constant of adsorption and q_e is the equilibrium capacity. The second-order kinetic model can be expressed by Eq. 4:

  

\begin{equation} \frac{1}{q_{\text{e}} - q_{\text{t}}} - \frac{1}{q_{\text{e}}} = k_{2}t \end{equation} (4)

The application of these two models to our experimental results, shown in Fig. 2a, revealed that the pseudo-first-order model (Eq. 3) provided a better fit with the experimental data than the second-order model (Eq. 4).

On the other hand, MWCNTs usually form aggregates in solution owing to their low hydrophobicity. Thus, we can expect that laccase adsorption on MWCNTs occurs in two steps: 1) external diffusion of laccase to the MWCNTs and 2) internal diffusion of laccase inside the MWCNT aggregates before contact with the MWCNT surface. Generally, in an intra-particle diffusion model, an intra-diffusion process with slower kinetics can be observed between external diffusion and the final equilibrium state as a result of the lower concentration gradient of laccase. Intra-particular diffusion usually becomes the limiting step in many adsorption procedures, especially in porous materials with pore sizes as large as those of the adsorbents. The possibility of intra-particular diffusion was explored using the diffusion model:²⁵

  

\begin{equation} q_{\text{t}} = k_{\text{dif}} \times t^{1/2} + \text{C} \end{equation} (5)

where C is the intercept and k_dif is the intra-particle diffusion rate constant. The intra-particle diffusion plot of q_t vs. t^1/2 revealed a two-phase process (Fig. 2b). The first phase represented the diffusion of laccase through the solution to the external surface of the MWCNTs and then to the surface through the boundary layer. In our study, the second phase represented the equilibrium of laccase adsorption. The plot of q_t vs. t^1/2 revealed that the intra-particle diffusion process was not observed, suggesting that the space size among the MWCNTs was wide enough to avoid deceleration of the diffusion of laccase.

3.3 Effect of temperature

Temperature is an important parameter for physical adsorption.²⁶ In the case of endothermic adsorption, such as chemisorption, the number of adsorbed molecules increases with increasing temperature because chemisorption involves the formation of chemical bonding, which requires activation energy. Conversely, in a physisorption process, the amount of adsorption decreases with increasing temperature because physisorption involves weak van der Waals forces, which weaken with increasing temperature.²⁷ The dependence of physisorption and chemisorption on temperature can be explained by the nature of the forces present to bind their particles. However, in the case of enzymes, the effect of temperature on adsorption is challenging to predict as it depends on several parameters, such as lateral interactions between adsorbed proteins, conformal changes, reorganization of adsorbed proteins, the denaturation temperature of the protein, and the composition of proteins. Czeslik et al. studied the effect of temperature on enzyme adsorption at the water/silica interface and found that adsorption of the enzyme was enhanced with increasing temperature, suggesting that adsorption is an endothermic process derived from entropic forces, which increases the reorientation mobility of enzyme segments.²⁸

As shown in Fig. 3, the amount of adsorbed laccase decreased with increasing temperature. Indeed, decreasing the temperature from 37 °C to 4 °C during adsorption resulted in up to a 50 % increase in the amount of adsorbed laccase. Therefore, the decrease in the amount of laccase on MWCNTs observed at the equilibrium state at high temperatures may be explained by the fact that the dynamics of laccase decrease at a higher temperature: the contact between laccase and the adsorbent is insufficient, leading to a decrease in adsorption efficiency. These results suggest that laccase adsorption on MWCNTs is an exothermic process that occurs via physical rather than chemical adsorption. These results agree with previous reports on the adsorption of laccase on MWCNTs and TiO₂ surfaces.^13,29

Figure 3.

Adsorption efficiency of laccase on MWCNTs at different temperatures. The error bars were evaluated by the Student t-distribution at 90 % confidence level.

3.4 Effect of ionic strength

Ionic strength, mainly driven by electrostatic interactions between proteins and the carrier surface, is known to have a significant impact on protein adsorption. An increase in the concentration of salt in the solvent results in a decrease in the number of adsorbed proteins. However, in the case of hydrophobic-interaction driven adsorption, the amount of adsorbed proteins was not affected by variations in the ionic strength of the solvent or even increased with increasing ionic strength of the solvent.^30–32

To investigate the effect of the ionic strength on laccase adsorption on MWCNTs, laccases dissolved in buffers of different ionic strength were used for the adsorption process. We measured the amount of these adsorbed laccase samples (at different ionic strengths) on pristine MWCNTs as well as on MWCNTs-NH₂, which are more hydrophilic than pristine MWCNT. As shown in Fig. 4, we observed an approximately 10 % decrease in the amount of adsorbed laccase on pristine MWCNTs when the ionic strength of the solution was increased from 50 to 500 mM. However, in case of MWCNTs-NH₂, increasing ionic strength from 50 to 500 mM decreased laccase adsorption by approximately 30 %. This result suggests that, in the case of pristine MWCNTs, the adsorption of laccase mainly results from hydrophobic interactions, which have low sensitivity to changes in the ionic strength of the solution. Conversely, in the case of MWCNTs-NH₂, the presence of amine groups on the surface of the MWCNTs makes the surface more electrically charged; this renders the contribution of electrostatic interactions to laccase adsorption more important and results in a heightened sensitivity to variations in ionic strength of the solution.

Figure 4.

Adsorption of laccase onto MWCNTs or MWCNTs-NH₂ at ionic strengths from 50 to 500 mM at 20 °C and pH 5.0. The error bars were evaluated by the Student t-distribution at 90 % confidence level.

3.5 Effect of pH

Protein adsorption on the solid surface is affected by the surface charges of the protein and surface, which can be altered by the pH of the solvent since the zeta potential and surface charge of the proteins vary with pH.^33,34 The adsorption process was studied at different pH values to optimize the effect of solvent pH (Fig. 5). For all pH values investigated, higher laccase adsorption was observed on MWCNTs than on MWCNTs-NH₂, and the percentage of adsorption linearly decreased with increasing pH. For both the materials, highest laccase adsorption was observed at pH 4.5, which is close to the isoelectric point of laccase (pH 4.2), where the charge of laccase is zero.¹⁵ This agrees with reported results that protein adsorption reaches a maximum when the pH of the solution is close to the isoelectric point of the protein.^14,15 The lower adsorption of laccase on MWCNTs-NH₂ suggests that the positively charged carbon nanotubes prevents the adsorption of laccase. The difference between MWCNT and MWCNTs-NH₂ become small when the zeta-potential of MWCNTs-NH₂ tend to move to zero near neutral pH.^14,15,35 This result indicates that even if the main driving force of laccase adsorption is hydrophobic interactions, electrostatic interactions between laccase and MWCNTs affect laccase adsorption.

Figure 5.

pH-dependent adsorption of laccase onto MWCNTs and MWCNTs-NH₂.

4. Conclusion

We investigated the kinetics and thermodynamics of laccase adsorption on MWCNTs. The adsorption of laccase on the surface of MWCNTs was explained by the Langmuir model with a pseudo-first-order kinetic model forming monolayer. The diffusion process of laccase inside the MWCNT matrix was not observed, suggesting that the matrix size among the MWCNTs was wide enough not to cause the deceleration of the diffusion. The hydrophobicity of MWCNTs renders their dispersion in solution incomplete and limits the amount of laccase adsorbed on MWCNT surfaces. We also observed that laccase adsorption on MWCNTs exhibits overshooting, which may result from the rearrangement and conformal changes in the adsorbed laccase during the process. Further, we demonstrated that laccase adsorption is an exothermic process resulting mainly from hydrophobic interactions between laccase and MWCNTs; however, our investigation of the effects of the pH and ionic strength of the solution indicates that the contribution of electrostatic interactions is not negligible. This analytical method can be widely applicable to a variety of enzymes as well as the support electrode, especially for designing potential biocatalyst for the molecular oxygen reduction reaction at the cathode in enzymatic biofuel cell applications.

Acknowledgments

The authors thank the following organizations for financial support: Campus France (Program Polonium 2019–2020), the French National Research Agency (BioWatts project ANR-15-CE05-0003-01, ImABic project ANR-16-CE19-0007-03), The Auvergne Rhones Alpes programs “cooperation international” and “Pack ambition international” (AZ), and JSPS KAKENHI Grant Number 18H01719 and 22K18912 (ST).

CRediT Authorship Contribution Statement

Awatef Ben Tahar: Data curation (Lead), Writing – original draft (Lead)

Alex L. Suherman: Investigation (Lead), Writing – original draft (Equal)

Abderrahim Boualam: Investigation (Equal), Writing – original draft (Supporting)

Seiya Tsujimura: Writing – review & editing (Lead)

Isao Shitanda: Writing – review & editing (Supporting)

Abdelkader Zebda: Funding acquisition (Lead), Methodology (Lead), Project administration (Lead), Resources (Lead), Supervision (Lead), Writing – review & editing (Lead)

Conflicts of Interest

There are no conflicts to declare.

Funding

Agence Nationale de la Recherche: ANR-15-CE05-0003-01

Agence Nationale de la Recherche: ANR-16-CE19-0007-03

Japan Society for the Promotion of Science: 22K18912

Région Auvergne-Rhône-Alpes: cooperation international

Footnotes

S. Tsujimura and I. Shitanda: ECSJ Active Members

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