Hosting Adamantane in the Substrate Pocket of Laccase: Direct Bioelectrocatalytic Reduction of O2 on Functionalized Carbon Nanotubes

Article abstract of DOI:10.1021/acscatal.6b00797

We report the efficient immobilization and orientation of laccase from Trametes versicolor on MWCNT electrodes using 1-pyrenebutyric acid adamantyl amide as a supramolecular linker. We demonstrate the ability of adamantane to specifically interact with the hydrophobic cavity of laccase, while pyrene interacts with MWCNT sidewalls by π-π interactions. Adamantane allows the oriented immobilization of laccases on MWCNT electrodes. Using an anthraquinone-modified pyrene derivative for comparison, adamantane-modified MWCNTs achieve the stable immobilization and orientation of a higher number of enzymes per surface units, as confirmed by electrochemistry, theoretical calculations, and quartz crystal microbalance experiments. Furthermore, the efficient direct electron transfer ensures bioelectrocatalytic oxygen reduction at high half-wave potential of 0.55 V vs SCE accompanied by no kinetic limitation by the heterogeneous electron transfer and maximum current densities of 2.4 mA cm^-2.

Full text of DOI:10.1021/acscatal.6b00797

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Article
Hosting adamantane in the substrate pocket of laccase: direct
bioelectrocatalytic reduction of O2 on functionalized carbon nanotubes
Noémie Lalaoui, Rolf David, Helene Jamet, Michael Holzinger, Alan Le Goff, and Serge Cosnier
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00797 • Publication Date (Web): 24 May 2016
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Page 1 of 8
ACS Catalysis
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Hosting adamantane in the substrate pocket of laccase: direct
bioelectrocatalytic reduction of O2 on functionalized carbon
nanotubes
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Noémie Lalaoui, Rolf David, Hélène Jamet, Michael Holzinger, Alan Le Goff* and Serge Cosnier
Univ. Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, France
CNRS, DCM UMR 5250, F-38000 Grenoble, France
ABSTRACT: We report the efficient immobilization and orientation of Laccase from Trametes versicolor on MWCNT
electrodes using 1-pyrenebutyric acid adamantyl amide as a supramolecular linker. We demonstrate the ability of ada-
mantane to specifically interact with the hydrophobic cavity of laccase while pyrene interacts with MWCNT sidewalls by
π-π interactions. Adamantane allows the oriented immobilization of laccases on MWCNT electrodes. Using anthraqui-
none-modified pyrene derivative for comparison, adamantane-modified MWCNTs achieve the stable immobilization and
orientation of a higher number of enzymes per surface units, as confirmed by electrochemistry, theoretical calculations
and Quartz Crystal Microbalance experiments. Furthermore, the efficient direct electron transfer ensures bioelectrocata-
lytic oxygen reduction at high half-wave potential of 0.55V vs SCE accompanied with no kinetic limitation by the hetero-
geneous electron transfer and maximum current densities of 2.4 mA cm-2.
KEYWORDS: carbon nanotubes, oxygen reduction, pyrene, multicopper oxidases, laccase
Laccases (E. C. 1.10.3.2) are blue multi-copper oxidases
that catalyze the four electron reduction of O2 to H2O
using phenolic compound as electron donor.1 Laccase
from Trametes versicolor reduces oxygen at low overpo-
tentials under weakly acidic conditions; and is therefore
of interest for technological applications, such as the
cathodic catalyst in enzymatic fuel cells (EFCs). One key
aspect in the design of next generation EFCs is the engi-
neering of enzyme wiring which combines excellent elec-
troactivity, stability and ease of production. Few years
ago, the crystal structure of laccase III from Trametes
versicolor (TvLAC) was solved.2,3 The four copper atoms in
each laccase are organized in two different sites: a trinu-
clear type 2/type 3 cluster (TNC) active site at which O2 is
reduced, and a mononuclear type 1 (T1) copper site, locat-
ed on the other side of the enzyme near a wide and hy-
drophobic pocket with the capacity to bind a wide range
of hydrophobic organic substrates of appropriate sizes.
Electrons are transferred one by one from the T1 site
through the protein to the TNC site where oxygen is re-
duced to water in a four electron process 1,4,5. Immobilized
laccases are able to transfer directly electron between the
T1 copper and conducting surfaces. This is a very attrac-
tive property for a fuel cell biocathode since, if the en-
zyme wiring is efficient, it avoids the need of soluble or
co-immobilized redox mediators. Many literature reports
have shown the great influence of surface functionalities
on enzyme immobilization and hence on the resulting
catalytic efficiency.6,7 Efficient orientation of laccases on
electrodes is of particular importance since it was demon-
strated that wiring the T1 copper center by direct electron
transfer (DET) with the electrode allows this enzyme to
work at a low overpotential, i.e only several tens of mVs
below the thermodynamic potential of the O2/H2O redox
couple8.
F. A. Armstrong et al. have proposed an alternative to
classical enzyme immobilization techniques by taking
advantage of the hydrophobic pocket to orientate and
immobilize the enzyme on the electrodes surfaces which
were covalently modified with anthracene or anthraqui-
none groups.9,10 This strategy relies on the functionaliza-
tion of the electrode surface using polycyclic aromatic
compounds that can interact with the hydrophobic pock-
et of laccase; composed of hydrophobic amino acids and
located near the T1 center; thus achieving both immobili-
zation and orientation of the enzyme. Carbon nanotube
(CNT)-based electrodes have been particularly employed
in the field of EFC due to their ability to wire enzyme
efficiently while providing a high specific surface area for
immobilizing a high amount of enzymes.5,11–13 Covalent
functionalization of CNTs with anthracene and anthra-
quinone groups has been achieved using different ap-
proaches: amide coupling with CNT defects,14 amination15
or diazonium chemistry.16–18 Non-covalent functionaliza-
tion of CNT is interesting compared to covalent modifica-
tion because the physical and electronic properties of the
CNTs are preserved. CNTs undergo strong π-π staking
interactions with π-extended planar molecules such as
pyrene.19 We have recently modified laccase mutants with
pyrene groups located near the T1 copper centre and
demonstrated the efficient DET between pyrene-modified
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laccases and CNTs.20 Pyrene derivatives bearing anthra-
laccases (figure 2). Adamantane is well-known to strongly
interact with β-CD, forming a host guest system.25 This
strategy allowed us in one hand to confirm the immobili-
zation of pyr-ADA on MWCNT surface, and in another
hand to mimick the attachment of TvLAC on the elec-
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quinone group have also been successfully used for the
immobilization of laccase.21 These anthraquinone-
modified pyrene molecules have demonstrated excellent
DET properties towards laccase. However, since anthra-
quinone can also strongly interact with CNT sidewalls, a
competition likely exists between interactions of anthra-
quinone groups with hydrophobic domain of laccase and
CNT sidewalls. For these reasons, this work proposes the
use of adamantane as a guest for laccase pocket while
exhibiting weak interactions with CNTs. In supramolecu-
lar chemistry, adamantane is well-known as an excellent
guest for hydrophobic cavity of β-cyclodextrins (β-CDs)22.
We have especially taken advantage of this host-guest
interactions to immobilize β-CD-modified proteins23–25 on
CNT-based electrodes. These studies have prompted us to
study the interaction of TvLAC with MWCNTs modified
by 1-pyrenebutyric acid adamantyl amide (pyr-ADA).
Electrochemistry, associated with kinetic simulations,
Quartz Crystal Microbalance (QCM-D) experiments and
theoretical calculations are aimed at characterizing ada-
mantane-laccase surface interactions and their influence
on bioelectrocatalysis.
26
trode. Indeed, these β-CD-AuNPs have approximately
the same size as compared to TvLAC and are better visu-
alized by an electron beam as the protein. In this ap-
proach, β-CD mimics the hydrophobic pocket of the en-
zyme. The ADA-MWCNT deposit was incubated in aque-
ous solutions of β-CD-AuNPs and subsequently rinsed
with water. Figure 2 unambiguously confirms the excel-
lent homogenous attachment of β-CD-AuNPs on ADA-
MWCNT film, as confirmed by the high density of spheri-
cal AuNPs at the surface of MWCNT sidewalls.
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RESULTS AND DISCUSSIONS
1. Characterization of the ADA-MWCNT/TvLAC
electrode
Glassy carbon electrodes (3 mm diameter) were function-
alized in several steps. First, MWCNTs were dispersed in
NMP until a homogeneous black suspension was ob-
tained. Then, a 5-µm-thick film of MWCNT was deposited
on the electrode surface by drop-casting of the MWCNT
suspension. Functionalization of MWCNTs through π-
stacking interactions was achieved by soaking the elec-
trode in 10 mM DMF solution of pyr-ADA, affording
ADA-MWCNTs (ADA-MWCNTs). Then, ADA-MWCNT
electrodes were incubated in the presence of solution of
TvLAC (5 mg mL-1 in Mc Ilvaine buffer pH5.0) (figure 1).
Figure 2. High-resolution SEM image of ADA-MWCNT after
incubation in aqueous β-CD-AuNP 0.5 mg mL-1 solution.
QCM-D experiments were performed in order to investi-
gate the immobilization of TvLACs on ADA-MWCNT
electrodes. Gold-coated piezoelectric quartz crystals were
modified with a pristine MWCNT film and ADA-MWCNT
film. A typical QCM-D profile for ADA-MWCNT film and
pristine MWCNT is displayed in figure 3A. The starting
baseline corresponds to the continuous flowing of the
buffer solution. After 20 min of signal stabilization, 300
µL of a TvLAC solution (5 mg mL-1 in Mc Ilvaine buffer
pH5.0) was injected during 6 min. The flow was stopped
during 1h, and then turned on to wash the functionalized
surface with Mc Ilvaine buffer pH 5.0. As soon as TvLAC
adsorption on MWCNT electrodes occurs, a frequency
decrease of 0.8 Hz and 5.3 Hz is observed for pristine
MWCNT and ADA-MWCNTs, respectively.
Figure 1. Schematic representation of the supramolecular
functionalization of MWCNT with pyr-ADA and TvLAC
To characterize the immobilization of pyr-ADA and its
ability to interact with hydrophobic cavities,, scanning
electron microscopy (SEM) of the surface of MWCNTs
was performed, using β-CD-modified gold nanoparticles
(β-CD-AuNPs, 3–5 nm in diameter) as surface probes of
the presence of adamantane and as nano-sized mimics of
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pmol cm-2. This in good agreement with previous studies
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where bilirubin oxidases have been immobilized on
MWCNT electrodes. A surface coverage of 5 pmol cm-2
has been measured based on non-catalytic redox system
corresponding to the T1 copper centre28.
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Pristine MWCNT and ADA-MWCNT electrodes modified
with TvLAC were then investigated by cyclic voltammetry
in Mc Ilvaine buffer pH 5.0. The cyclic voltammograms
(CVs) recorded under argon and oxygen-saturated solu-
tions are presented in Figure 4. As for QCM-D experi-
ments, the biocathodes were compared to pristine
MWCNTs electrodes similarly incubated with TvLAC.
While TvLAC adsorbed on pristine MWCNT exhibits
maximum current densities of 0.6 (+/-0.2) mA cm-2 under
oxygen, TvLAC immobilized on ADA-MWCNTs exhibit a
plateau current at 2.2 (+/-0.4) mA cm-2 accompanied with
a half-wave potential of 0.55 (+/-0.2)V.
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Figure 3. (A) QCM-D profile (shifts in resonant frequency
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(black curve) and in dissipation (red curve) vs. time) for the
immobilization of TvLACs on ADA-MWCNTs (full line) and
pristine MWCNTs (dotted line) (13th overtone data) before
and (a) after injection of 5 mg mL-1 TvLAC solution in Mc
Ilvaine buffer pH 5.0 for 6 min, (b) stop of the flow for 1 h
and (c) restart of the flow of Mc Ilvaine buffer ; (B) Plot of
TvLAC mass uptake vs. time calculated from the Sauerbray
equation (1) for the 3, 5, 7, 9, 11 and 13th overtone
For pristine MWCNTs, as a result of the TvLAC injection
stop after 6 min, an increase of the frequency is observed
until the baseline signal is recovered. On the contrary, the
stable interactions between TvLAC and ADA-MWCNT
results in a stable frequency signal, even when Mc-Ilvaine
buffer is passed in the QCM-D cell after 1h. In parallel, the
increase of the dissipation factor in the case of ADA-
MWCNT reflects a decrease in the rigidity of the MWCNT
surface, which likely arises from the stable immobiliza-
tion of enzymes. Figure 3B displays the plot of the mass of
TvLAC adsorbed at the surface of the ADA-MWCNT film
as a function of time. The mass uptake of TvLAC was
estimated using the Sauerbrey equation 1 for different
overtones n = 3, 5, 7, 9, 11 and 13th
Figure 4.Cyclic voltammograms (CVs) of TvLAC-
functionalized MWCNT electrodes in stirred oxygen-
saturated solution for (a) pristine MWCNT and (b) ADA-
MWCNT electrodes ; (c, dashed line) CV of the TvLAC-
functionalized MWCNT electrode in argon-saturated solu-
tion (Mc Ilvaine buffer pH 5, v= 0.01V.s-1, RT). CVs were re-
produced on at least 3 electrodes.
This corresponds to 4 fold increase of current density as
compared with electrodes using TvLAC non-specifically
adsorbed on MWCNTs, which is in good agreement with
QCM-D experiments. A half-wave potential of 0.55 V is in
good agreement with an efficient direct wiring of TvLAC
via the T1 copper centre.5 Besides, the addition of 2,2'-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS,
to the solution did not provoke significant increase in the
electrocatalytic current density for ADA-MWCNT. This
confirms that immobilized TvLAC on ADA-MWCNT film
are well connected to the electrode.15,28 Furthermore,
addition of ABTS did not provoke any decrease of current
density which might have been caused by a competitive
interaction between ABTS and adamantane towards the
∆ꢀ ꢁ ꢂꢃ∆ꢄ /ꢆ
(1),
ꢅ
Where C is the mass sensitivity C= 17.7 ng cm-2 Hz-1 at f1 =
5 MHz and n is the accordant overtone number. Curves
for each overtone show an average mass of immobilized
TvLAC of 80 ng cm-2 for ADA-MWCNT. Considering a
50% degree of hydration for the enzyme layer,27 this
would corresponds to a TvLAC surface density of 0.6
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Figure 5. Experimental (black) and simulated (red) CV scan
laccase substrate pocket. Control experiments in the same
buffer conditions have been performed on pristine
MWCNTs without any enzyme modification. MWCNTs
shows poor ORR activity, starting at low potentials below
0 V vs SCE.
1
2
3
4
for TvLAC-modified (A) MWCNT, (B) AQ-MWCNT and (C)
ADA-MWCNT electrodes under oxygen (Mc Ilvaine buffer
pH 5, v= 0.01V.s-1, RT). Background was subtracted to remove
the MWCNT film capacitive current contribution.
5
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Table 1. Fitting parameters according equation 1 for
TvLAC functionalized MWCNT electrodes.
The long-term stability of the enzyme electrodes were
also investigated, by performing one-hour discharge at 0.2
V every day during one month (figure S1). TvLACs immo-
bilized on ADA-MWCNTs show excellent activity over
weeks, still achieving 66 % of their bioelectrocatalytic
activity after one month, namely 1.32 mA cm-2. On the
contrary, non-specifically adsorbed TvLACs exhibits a
rapid decrease in terms of catalytic currents below 5% of
the initial value after 20 days. These experiments corrobo-
rates QCM-D experiments which also underline the supe-
rior stability of adamantane-modified MWCNT electrodes
towards immobilization of TvLACs.
E0
jlim
kcat/k0
βd0
R2
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MWCNT
0.52
0.52
0.55
0.52 0.005*
0.74 0.4
15
-
0.999
0.998
0.998
AQ-
MWCNT
ADA-
2.13
-
-
MWCNT
max
*This value corresponds to the ratio kcat/ko
tion 2
from equa-
2. Electrochemical Modeling
For non-specifically adsorbed TvLACs, the electrocatalytic
Electrochemical properties of ADA-MWCNT bioelec-
trodes were compared with those of previously-described
waveshape is well-modeled using equation 2 (derived
31
from equation from ref ),
MWCNTs
modified
with
bis(2-
anthraquinonyl)aminomethyl]pyrene (pyr-(AQ)2)29 AQ-
ꢇꢈꢉꢊ ꢎꢏ ꢂ ꢎꢐ
ꢋꢌꢍ 1 ꢑ ꢎꢏ
ꢓꢎꢏ∝ ꢑ 1 ꢑ ꢎꢏ
,
ꢇ ꢁ
ꢒꢆ
ꢚ2ꢛ
MWCNT, by modelization of the electrocatalytic
waveshapes. F. A. Armstrong and colleagues have defined
a model describing reversible electrocatalytic properties
of redox enzyme films, taking into account the different
orientations adopted by enzymes on the electrode sur-
face.30–32 These different orientations result in the disper-
sion of ET rates along with multiple active-site/electrode
distances. We have recently shown that this model could
describe well the different orientations adopted by biliru-
ꢔꢕꢖꢗ
∝
ꢚ
ꢛ
ꢎꢏ ꢑ 1 ꢑ ꢎꢏ expꢚꢂꢋꢌꢍꢛ
ꢔꢘꢊꢖꢙ
Where e1
=
exp(nF/RT)(E-E°Cu(T1)), e2
=
exp(-
4F/RT)(E°Cu(T1)-E°O2/H2O), kcat is the catalytic rate constant
max
for O2 reduction. k0
is the heterogeneous ET rate con-
stant at the minimal distance d0. The product βd0 is an ET
tunneling parameter. α is the transfer coefficient which is
set at 0.5. E°O2/H20 = 0.69 V vs SCE at pH 5. In this equa-
tion, it is assumed that catalytic rate constant for oxygen
reduction is largely higher as compared to catalytic rate
constant for water oxidation.31 Furthermore, all models
were well-fitted using n= 1. This is expected from the fact
that the rate-determining step is the electron transfer to
the T1 copper centre.20,28 For pristine MWCNTs, a βd0
value of 15 indicates a dispersion of ET rates arising from
different statistical orientations of TvLAC. By making the
approximation that the decay constant β for the dispersed
ET rates is similar to decay constant the decay constant of
bin oxidases and TvLACs on functionalized MWCNTs20,28
.
This model was used to investigate the influence of the
modified MWCNT surfaces on the electrocatalytic activity
and, by consequence, on the orientation of TvLACs. Ex-
periments were performed under stirring or at rotating-
disk electrodes, affording kinetically-controlled electroca-
talysis. Figure 5 displays the experimental and simulated
electrocatalytic waves for pristine MWCNTs, AQ-
MWCNTs and ADA-MWCNTs. Table 1 displays the fitting
parameters for the three electrocatalytic behavior from
figure 5.
33
1.1 Å-1 measured in azurin, this would mean that ET
rates vary along an average distance of 14 Å. This is closed
to the values which have been measured for bilirubin
oxidases from Myrothecium verrucaria immobilized on
similar pristine MWCNT electrodes.28 On the contrary of
unspecific adsorption of TvLACs on pristine MWCNTs,
AQ-MWCNT electrodes follows a simpler kinetic accord-
ing to equation 3 with no dispersion of ET rates.31
ꢜ ꢞꢜ
ꢝ
ꢟ
ꢇ ꢁ ꢇꢈꢉꢊ
(3)
ꢡ
ꢢꢣꢤ
∝
ꢏꢠꢜ ꢠ
ꢜ
ꢝ
ꢝ
ꢡ
ꢥ
Where k0 is the heterogeneous ET rate constant. The fact
that this equation fits well for the AQ-MWCNT setup
indicates negligible dispersion of ET rates, which con-
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talene16,17 or naphtoquinone,39 which exhibit strong inter-
firms that the enzyme is properly oriented towards the T1
1
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3
4
5
6
7
8
at the surface of CNTs, as it has been suggested previous-
ly. 18,21Furthermore, a kcat/k0 ratio of 0.4 indicates that ko
value is approximatively twice the value of kcat. Finally,
the electrocatalytic waveshape of ADA-MWCNT elec-
trodes indicates that electrocatalysis is not kinetically-
limited by DET. In this case, electrocatalytic currents
measured at each potentials is only limited by the catalyt-
ic turnover of TvLAC and can be defined according to a
actions with laccase pocket. Root mean square deviations
along trajectories were computed for inclusion complexes
obtained with adamantane and anthraquinone using the
initial structure as reference (Fig. S2). After 2.5 ns, a stable
position was found for both molecules, with a rmsd value
around 1.6Å.The binding modes are depicted in Figure 6.
Both molecules penetrate in the substrate cavity. For
anthraquinone, value of the average distance between the
oxygen atom of one carbonyl group and the T1-copper
atom is equal to 7.1 Å. Two hot spot residues interact
strongly with the molecule. One histidine bound to the
copper atom of the T1 site, the histidine 395 which forms
a hydrogen bond with a carbonyl group of anthraquinone,
and the phenylalanine 162 which orientates the position
of anthraquinone through π-stacking interactions. Ada-
mantane penetrates less in the cavity, the average dis-
tance between a hydrogen atom of the molecule and the
T1-copper atom is 11.9 Å. However, as for anthraquinone,
this position is stable and is due to two residues, the same
phenylalanine 162, which interacts with anthraquinone,
but also a second phenylalanine, the phenylalanine 332.
CH/π interactions are formed between these residues and
adamantane. CH/π interactions are formed between these
residues and adamantane (figure S2).
34
9
simple Nernst equilibrium. This type of highly efficient
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wiring of laccases has already been demonstrated for
Laccases from Trametes hirsuta or Trametes sp C30 im-
20,35
mobilized on AuNPs.
In this case, heterogeneous ET
rate is no longer limiting and electrocatalytic activity is
only limited by the enzyme’s surface coverage and catalyt-
ic activity. Only two parameters are thus to take into
account in the modeling of curve c from Figure 5. This
implies that the presence of pyr-ADA is responsible for
the absence of ET rate dispersion, a favorable orientation
of TvLACs for DET via the T1 copper centre and a high k0
value over kcat. When comparing jlim values of 2.13 and 0.52
mA cm-2 for ADA-MWCNTs and pristine MWCNTs re-
spectively, it is noteworthy that this 4-fold increase in jlim
value correlates with the surface coverage of TvLAC on
MWCNT and ADA-MWCNT electrodes measured by
QCM-D, which shows a 6-fold frequency decrease for
ADA-MWCNTs as compared to MWCNTs. More im-
portantly, the favorable orientation of the enzyme plays a
major role on the dispersion of ET rates and the behav-
iour of TvLAC at low driving force, i.e near the onset po-
tential. For instance, at 0.5 V vs SCE, electrocatalytic cur-
rents of -1.9 mA cm-2 and - 0.14 mA cm-2 were measured
for ADA-MWCNTs and MWCNT respectively, corre-
sponding to a 14-fold increase. According TvLAC surface
overage measured by QCM-D, a kcat value of 9 103 s-1 was
estimated for bioelectrocatalytic oxygen reduction. For
comparison, kcat values of 2.4 103 s-1 has been measured for
oriented bilirubin oxidases from Myrothecium verrucaria
on porphyrin-modified MWCNTs. 28
Molecular mechanics dynamics were done using explicit
water molecules to model the solvent before performing
MM-GBSA calculations.40 In this approach, counterions
and water molecules are removed on structures obtained
during the MD trajectories after the time of equilibration.
Inclusion of solvatation effects using the generalized born
model is combined to surface area and MM calculations
to estimate the binding free energies of each complex. In
agreement with the description of the interactions, the
free binding energy of anthraquinone, equals to -15.4 (+/-
1.8) kcal mol-1, is stronger than that of adamantane. Nev-
ertheless, the value obtained for adamantane, -7.8 (+/-1.5)
kcal mol-1, illustrates the positive interaction of this mole-
cule with the hydrophobic cavity of TvLAC.
3. Theoretical modeling
To investigate interactions between adamantane and
TvLAC, the molecule was docked at the enzyme using
36
Autodock program
as described under computational
methods in the Supporting information. The lowest and
the most recurrent scoring pose of all the docking runs
was chosen at the binding mode. Then, molecular me-
chanics (MM) dynamics were performed on this docked
position to take into account the flexibility of the protein
and to study the stability of the binding mode in solution.
Explicit water molecules are used to modelize the solvent
(see computational methods in SI).
For comparison, calculations were also conducted on
anthraquinone, which is known as a substrate and hydro-
phobic anchor of TvLAC.9,14,29,37 Anthraquinone was cho-
sen as a good representative of the family of polycyclic
compounds such as anthracene9,14,38, pyrene,26 naph-
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anthraquinone with laccase and with CNT sidewalls. This
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is the reason why adamantane interacts less strongly than
anthraquinone but provide a higher density of immobi-
lized enzyme at the surface of MWCNTs. Theoretical
calculations can let us envision the study of a library of
molecules with a stronger ability to interact with laccase,
as compared to adamantane. Furthermore, the integra-
tion of these adamantane-modified CNTs at the cathode
of enzymatic fuel cells is currently underway.
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ASSOCIATED CONTENT
SUPPORTING INFORMATION
Figure S1, S2, S3, Table S1, S2, experimental part and compu-
tational part. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* alan.le-goff@univ-grenoble-alpes.fr
Author Contributions
The manuscript was written through contributions of all
authors. / All authors have given approval to the final version
of the manuscript.
ACKNOWLEDGMENT
The authors wish to acknowledge the support from the
platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG)
and from the LabEx ARCANE (ANR-11-LABX-0003-01).
The authors also thank the ANR Investissements d'avenir-
Nanobiotechnologies 10-IANN-0-02 program for financial
support. They gratefully acknowledge funding from the
Agence Nationale de la Recherche through the project
CAROUCELL (ANR-13-BIOME-0003-02). They also thank
the GDR CNRS 3540 “Biopiles” for partial financial sup-
port. Electron microscopy was performed at the CMTC
characterization platform of Grenoble INP supported by
the Centre of Excellence of Multifunctional Architectured
Materials "CEMAM" n°AN-10-LABX-44-01 funded by the
"Investments for the Future" Program. They would like to
thank ZEISS for valuable support and discussion in oper-
ating GeminiSEM 500. The authors thank prof. Reynaldo
Villalonga for providing β-cyclodextrin-modified gold
nanoparticles. Hugues Bonnet and Liliane Guerente are
acknowledged for valuable help and discussions on QCM-
D experiments.
Figure 6. Binding modes obtained for (A) anthraquinone-
TvLAC and (B) adamantane-TvLAC complexes in solution.
Water molecules are removed to simplify the pictures. Cop-
per atoms are illustrated in magenta; carbon, oxygen and
nitrogen atoms of hot spot residus in cyan, red and blue and
carbon atoms of adamantane and anthraquinone in green
respectively.
CONCLUSION
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