Comparison of bioelectrocatalysis at Trichaptum abietinum and Trametes hirsuta laccase modified electrodes

Electrochimica Acta 130 (2014) 141–147

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Comparison of bioelectrocatalysis at Trichaptum abietinum and Trametes hirsuta laccase modified electrodes Marius Dagys a,∗ , Peter Lamberg b , Sergey Shleev b , Gediminas Niaura a,1 , Irina Bachmatova a , Liucija Marcinkeviciene a , Rolandas Meskys a , Juozas Kulys a , Thomas Arnebrant b , Tautgirdas Ruzgas b a b

Institute of Biochemistry, Vilnius University, Mokslininku˛ str. 12, LT-08662, Vilnius, Lithuania Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE-20506 Malmö, Sweden

a r t i c l e

i n f o

Article history: Received 2 December 2013 Received in revised form 26 February 2014 Accepted 3 March 2014 Available online 15 March 2014 Keywords: Electron transfer Bioelectrocatalysis Oxygen reduction Gold nanoparticle Laccase

a b s t r a c t Bioelectrocatalytic reduction of oxygen to water at electrodes modified with gold nanoparticles and a new laccase from Trichaptum abietinum (TaLc) was studied. The bioelectrocatalytic current was found to be much higher at TaLc modified electrodes than at similarly prepared electrodes modified with a broadly used laccase from Trametes hirsuta (ThLc). To explain this difference the bioelectrocatalysis was described in terms of kinetic rate constants based on simultaneous cyclic voltammetry and quartz crystal microbalance measurements. From analysis of the rate constants both laccases appeared to possess similar rates (k0 ) of direct electron transfer. However, the enzyme turnover (kcat ) was about three-fold higher for gold nanoparticle bound TaLc than for ThLc, calculated using surface concentration of enzyme established by QCM-D. Near reversible potential-induced reorientation of adsorbed proteins was observed by surface enhanced Raman spectroscopy. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Design of bioelectrochemical devices, e.g. biosensors and biofuel cells (BFCs), based on direct electron transfer (DET) reactions between redox enzymes and conducting electrode materials is very attractive due to the conceptual simplicity of their operation mechanism [1–3]. Theoretically, the DET principle allows the construction of biosensors which are minimally affected by interfering reactions as well as BFCs with the highest cell potential since redox mediators are not required to shuttle electrons between the redox centre of enzymes and the surface of the electrodes. DET based bioelectrocatalysis with laccases was first discovered as early as in 1978 [4] and later for many other oxidoreductases [5]. From the most recent studies of DET [5,6] it can be concluded that DET-based bioelectrocatalysis is more easily realised at nanostructured electrodes. This observation probably cannot be generalised since there are a number of examples where rapid DET at planar electrodes has been reported, specifically by addressing the enzyme orientation at the electrode surface [7,8].

∗ Corresponding author. Tel.: +370 5 272 91 76; fax: +370 5 272 91 96. E-mail address: [email protected] (M. Dagys). 1 ISE member http://dx.doi.org/10.1016/j.electacta.2014.03.014 0013-4686/© 2014 Elsevier Ltd. All rights reserved.

Additionally, exploiting nanomaterials for constructing the DETbased biosensor or BFCs provides an opportunity to create a variety of 3D nanostructures, e.g. hierarchical carbon materials [9] and metallised nanostructured silica [10]. It has been demonstrated that even a simple drop-casted and dried gold nanoparticle (AuNP) dispersion results in 3D structure, which impregnated with an enzyme enabled superior bioelectrocatalysis, specifically, high current density and superior operational stability [11,12]. These findings suggest that a combination of redox enzymes and nanomaterials can result in efficient and possibly commercially competitive bioelectrochemical devices. Taking this into account, finding new enzymes for DET-based bioelectrocatalysis is highly motivated. In this work we report on a DET-based bioelectrocatalytic reduction of oxygen to water (Eq. 1) by a new laccase from Trichaptum abietinum (TaLc) at AuNP-modified electrodes. TaLc enables much higher bioelectrocatalytic current if compared to a well-studied and broadly used laccase from Trametes hirsuta (ThLc) [6]. The mechanisms of homogeneous catalysis and bioelectrocatalysis (Fig. 1) of these two laccases are considered to be principally equal and can be summarised by 4-electron reduction of di-oxygen to water (Eq. 1). The electrons in the homogeneous reaction are received from soluble reducing compounds, e.g., phenol, while in heterogeneous reaction from the electrode by direct electron transfer (DET). Lc

O2 + 4e + 4H + −→2H2 O

(1)

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with the extinction efficiency vs. wavelength generated by the MiePlot software, which can be found as a freeware at http://www.philiplaven.com/mieplot.htm. 2.2. Enzymes

Fig. 1. Schematic representation of bioelectrocatalytic reduction of oxygen to water based on DET between a laccase molecule and the AuNP modified planar gold electrode. The electrons flow from the planar electrode (AuQCM-D), through the gold nanoparticle (AuNP), to the T1 copper site and finally to the T2/T3 copper cluster where oxygen is reduced to water. The maximum bioelectrocatalytic current density (jmax ) depends on the activity of enzyme (kcat ). The rate of DET is characterised by the standard rate constant, k0 , which determines current dependence on applied potential, E. Other notations are: kfDET and kbDET are forward and backward rate constants for direct electron transfer between the T1 site and AuNP; ˛ is the transfer coefficient. A full mathematical description of electrode current can be found in [13].

To understand the reasons for superior bioelectrocatalysis achieved with TaLc combined electrochemical and quartz crystal microbalance with dissipation (QCM-D) measurements have been carried out. The experiments revealed that both laccases show very similar heterogeneous DET rate (k0 ), however, the catalytic activity (kcat ) for TaLc bound to AuNP-modified electrodes was approximately three-fold higher than for ThLc bound to AuNPs. Surface enhanced Raman spectroscopy (SERS) has been employed for molecular level characterisation of adsorption of enzymes on AuNPs. 2. Experimental 2.1. Chemicals 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Na2 HPO4 . 2H2 0, NaCl, Na2 SO4 , HAuCl4 × 3H2 0, NaCH3 COO, poly-L-lysine (PLL) and citric acid monohydrate were purchased from Sigma (St. Louis, MO, USA). H2 SO4 and NaF were obtained from Merck (Darmstadt, Germany). Trisodium citrate-2-hydrate was purchased from Riedel-de Haën, Seelze, Germany. All chemicals were of analytical grade. Buffers and all other solutions were prepared using deionised water (18 M. cm) purified with a PURELAB UHQ II system from ELGA Labwater (High Wycombe, UK). The majority of the measurements were performed at room temperature using phosphate-citrate buffer (PCB), containing 50 mM Na2 HPO4 solution with 0.1 M Na2 SO4 , pH 4.0 (adjusted by addition of citric acid), unless stated otherwise. Gold nanoparticles (AuNPs) were synthesised from HAuCl4 salt by using citrate as a reducing agent [14–17]. Their diameter was determined by dynamic light scattering using a Nicomp 380 ZLS unit (Santa Barbara, California). The concentration of AuNPs in dispersion was determined by comparing (overlapping) the optical absorption spectra of their colloidal dispersions

The solution of homogeneous fungal laccase from Trametes hirsuta basidiomycete (EC 1.10.3.2), with 70 kDa molecular weight including 12% carbohydrate and a pI 4.2, was prepared as described in [18]. The concentration of the ThLc in the stock solution was 18 mg ml−1 . A homogeneous preparation was stored frozen in 0.1 M phosphate buffer, pH 6.5 at–18 ◦ C. The solution of homogenous fungal laccase from Trichaptum abietinum basidiomycete with a molecular weight of 51 ± 2.5 kDa and a pI 4.7, was prepared as described in [19]. The concentration of the TaLc in the stock solution was 21 mg ml−1 . A homogeneous preparation was stored frozen in 0.01 M phosphate buffer, pH 7.0, at–18 ◦ C. Activities of laccases were assessed by examining the oxidation of ABTS to its cation radical (ABTS+. ) at 420 nm (␧420 = 36000 M−1 cm−1 ). The reaction mixture contained 5 mM ABTS in PCB and a suitable amount of enzyme. Enzyme turnover for ThLc and TaLc were 220 s−1 and 390 s−1 , respectively, where 1 s−1 of turnover is defined as the amount (␮mol) of ABTS+. formed by 1 ␮mol of enzyme in one second at 23 ◦ C. Before the experiment the enzymes were diluted by PCB to the required concentration and kept at 4–8 ◦ C. 2.3. Electrode preparation To investigate bioelectrocatalytic reactions of laccases adsorbed on AuNPs several electrode designs have been used. Specifically: (1) quartz crystal sensor with planar gold layer, AuQCM-D , i.e., QCMD sensor; (2) silicon wafer covered with evaporated 200 nm Au layer for ellipsometric measurements; planar surfaces of these electrodes were plasma cleaned (Harrick, model PDC-32G) for 10 min at the highest plasma intensity setting; (3) gold rod press-fitted in Teflon for SERS measurements. Electrical potentials applied to the electrodes are reported vs SHE in this paper. 2.4. Simultaneous QCM-D and cyclic voltammetry measurements To characterise bioelectrocatalysis in terms of reaction rate constants (see Fig. 1) QCM-D and cyclic voltammetry (CV) measurements were run simultaneously. The aim with these experiments was to form a monolayer of AuNPs on the planar gold surface of the QCM-D sensor, subsequently adsorb laccase, and register the oxygen bioelectroreduction current by running a CV measurement. For QCM-D sensor modification the following solutions were pumped through the QCM-D cell containing the sensor: 1) 0.002 wt% poly-Llysine (PLL) solution in water, 2) 50 ␮g ml−1 laccase solution in PCB, 3) a dispersion of AuNPs in water (for faster AuNP adsorption at the AuQCM-D -PLL-Lc surface the AuNP dispersion with NP diameter of 30 - 40 nm contained 20 mM NaCl, 50–60 nm–10 mM NaCl, larger than 80 nm had no NaCl added.) and 4) 50 ␮g ml−1 laccase solution again as in step two. The QCM-D technique enabled mass measurements, specifically, the mass of AuNPs as well as the mass of laccase bound at the electrode surface. At the end of each electrode modification procedure the bioelectrocatalytic current was registered for each electrode by CV measurements. Knowing the mass of the adsorbed AuNPs, the mass of laccase (from QCM-D), and the electrode current at different potentials (from the CV) enabled the calculation of the standard heterogeneous electron transfer rate (standard rate of DET), k0 , between the AuNP and laccase, and the apparent enzyme turnover rate, kcat . All QCM-D measurements were made at a temperature of 23 ± 0.02 ◦ C controlled by Peltier element included in QWEM401 module. From previous studies it is known [20] that only the laccase adsorbed at AuNPs enables DET-based

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Fig. 2. Voltamperometric analysis made by using AuNP - modified QCM-D gold sensor electrode with adsorbed TaLc (A) or ThLc (B). The curves represent a cathodic sweep process modelled as described in [13], where the background current recorded with 5 mM NaF present in the buffer solution is subtracted. AuNPs with 20 (red), 37 (green) and 95 nm (blue) diameter have been used. Buffer solution: 50 mM Na2 HPO4 , 0.1 M Na2 SO4 , pH 4.0, adjusted with citric acid; potential scan rate is 5 mV s−1 .

bioelectrocatalytic current. Despite these previous investigations all necessary control CV measurements were performed anyway, confirming already published features of the Lc-AuNP system. A theoretical description of the current-potential dependency and fitting procedure has been previously published [13,20]. The adsorbed laccase mass was calculated using Qtools software from Q-Sense (Västra Frölunda, Sweden). It should be noted that QCMD measurements of the surface bound mass include the mass of bound water [21]. To estimate the amount of water bound in the laccase layer on gold surface the adsorption process of laccase on planar gold was monitored using QCM-D and null ellipsometry. Ellipsometry provides an estimation of the “dry mass” of the adsorbed film. The amount of coupled water was calculated as a difference of the mass of the laccase layer adsorbed on gold determined by QCM-D minus the laccase mass determined by ellipsometry (data are not shown). 2.5. Surface enhanced Raman spectroscopic measurements To characterise molecular AuNP-laccase interactions nearinfrared SERS spectra were recorded using a Raman spectrometer RamanFlex 400 (Perkin Elmer, Inc., USA) equipped with a thermoelectrically cooled (−50 ◦ C) CCD camera, a diode laser for excitation (785 nm, 30 mW power was focussed to a 200 ␮m diameter spot on the electrode), and a fibre - optic cable for directing the excited and scattered beams. The 180o scattering geometry was employed. Raman spectra were recorded by using 10 s integration time and by summing 30 scans. Spectroelectrochemical measurements were carried out in a cylinder-shaped three electrode moving cell, arranged with a working electrode, a platinum wire as a counter electrode, and an Ag/AgCl reference electrode. During the experiment solution was continuously bubbled with ultra-pure Ar gas to remove dissolved oxygen. The working electrode was placed at approx. 3 mm distance from the cell window. In order to reduce photo- and thermoeffects, the cell together with the electrodes were moved linearly with respect to the laser beam at a rate of about 15–25 mm s−1 [22,23]. The Raman frequencies were calibrated using a standard spectrum of polystyrene (ASTM E 1840). Intensities were calibrated by NIST intensity standard (SRM 2241). 3. Results and discussion From previous studies [6] it is known that the laccase from Trametes hirsuta (ThLc) adsorbed at AuNPs shows a considerable DET-based bioelectrocatalytic oxygen reduction to water [2]. The bioelectrocatalysis at a 20 nm diameter AuNP-ThLc bionanostructure has been more carefully studied at the most favourable condition and was characterised by a standard DET constant k0 = 6

s−1 and catalytic rate constant kcat = 13 s−1 [20]. In this work we investigated another laccase for bioelectroreduction of oxygen, more specifically, the enzyme from Trichaptum abietinum (TaLc). It can immediately be noticed (Fig. 2) that the bioelectrocatalytic current was much higher at gold nanoparticles modified by this laccase (denoted as AuNP-TaLc) than at AuNP-ThLc (Fig. 2B, compare the current at an applied voltage of 0.6 V). The removal of oxygen from the solution by N2 bubbling or inhibition of laccase by NaF almost completely diminished the current confirming that the current was due to DET-based laccase-catalysed electroreduction of oxygen (Eq. 1). However, even though the current at AuNP-TaLc modified electrodes (Fig. 2A) was much higher than for AuNP-ThLc, the reasons for the improved bioelectrocatalysis could not easily be pointed out. From Fig. 2 it seems that bioelectrocatalysis depends on the size of NPs used for the electrode modification. The dependence of bioelectrocatalysis vs NP size might be caused by a difference in the amount of AuNP binding to the electrode surface (e.g., the surface density of NPs), the amount of adsorbed enzyme (), a difference in the activity (kcat ) of the surface bound enzyme and the difference in rate of DET (k0 ) between the enzyme and the electrode surface. To establish the reasons for improved bioelectrocatalysis when using TaLc for electrode modification, combined QCM-D and electrochemistry data were processed to calculate k0 and kcat . The results are summarised in Figs. 3 and 4. From Fig. 3 it seems that amount of adsorbed enzyme did not depend on AuNP diameter and that in average adsorbed molar amount of TaLc was 1.7 ± 0.7 higher than ThLc. From Fig. 4 it seems that the highest rate constants were achieved at 30–50 nm diameter AuNPs, however, high scattering in the experimental data values did not allow a more firm conclusion to be drawn on the dependence of bioelectrocatalysis on the size of AuNPs. Averaging bioelectrocatalytic constants obtained at 20–100 nm AuNPs for each laccase gave the following results: DETbased bioelectrocatalysis by TaLc can be characterised by k0 = 5 ± 4 s−1 and a catalytic rate constant kcat = 17 ± 15 s−1 while bioelectrocatalysis with ThLc at the same conditions can be characterised by k0 = 3 ± 4 s−1 and a catalytic rate constant kcat = 5 ± 7 s−1 . Taking into account high standard deviations for the corresponding constants found for TaLc and ThLc it seems that the laccases cannot be distinguished in terms of the rate constants. However, if the ratio of k0 for TaLc vs ThLc at the same AuNP size and similarly the ratio for kcat were calculated an obvious difference should appear. The ratio for k0 was found to be equal to 1.0 ± 0.8 and the ratio for kcat was equal to 2.9 ± 1.0. These values indicated that both laccases showed a very similar rate of DET (k0 ), but the catalytic rate kcat was approximately three-fold higher for Trichaptum abietinum laccase bound to AuNP compared to Trametes hirsuta laccase. One of the possible reasons for this difference could be that Trichaptum abietinum laccase, being a smaller enzyme, was less conformationally changed at the

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Fig. 3. Amount of laccase adsorbed on QCM-D gold sensor surface modified with AuNPs. The data points represent molar surface concentration (pmol cm−2 , where cm−2 is a geometric surface area of QCM-D sensor) of ,,dry” TaLc (red) and ThLc (blue). The QCM-D sensor was modified with AuNPs of different diameter and the total amount of Lc adsorbed on AuNPs was determined by QCM-D measurements. Red and blue lines represent the mean surface concentration for TaLc and ThLc, respectively. On average, adsorbed mass of TaLc was 1.7 ± 0.7 fold higher than that of ThLc.

AuNP surface. Commenting on the dependence of bioelectrocatalysis on the size of NPs it could be stated that such dependencies have been studied by others with no general conclusion. In some cases higher bioelectrocatalytic currents were found at smaller nanoparticles [20,24,25] in some cases on larger nanoparticles [26]. Our current understanding is that these dependencies are not just the consequence of enzyme-nanoparticle interaction, but depend also on the 3D structures which are formed by NPs as well as other components of the bioelectrocatalytic layers, e.g., thiols or polyelectrolytes used to “glue” nanoparticles. It should be noted that the above summarised calculations of constants were done excluding the mass of coupled water from the mass of the laccase layer bound to gold. For this purpose combined ellipsometric and QCM-D measurements were conducted, revealing that adsorbed TaLc layer contains 68 ± 2 wt% of water, whereas the layer of ThLc contains 84 ± 5 wt%. Higher amount of water in the layer of surface bound ThLc could be explained by its presumably higher degree of glycosylation, as indicated by its higher mass. TaLc has 27% lower mass, but the degree of its glycosylation is unknown. To characterise Lc-AuNP interactions more precisely and to understand the nature of DET reactions at the molecular level surface enhance Raman spectroscopy (SERS) was exploited. The T1 copper centre usually exhibits an intense resonance Raman

Fig. 5. Raman spectrum of ThLc dissolved in buffer solution. Raman bands of phosphate anions present in buffer solution are indicated by asterisks. Measurement conditions: excitation wavelength – 785 nm, laser power at the sample – 100 mW, total integration time – 2000 s.

line near 415–430 cm−1 [27] when excited with red laser, e.g. at the wavelength of 647 nm. Pre-resonance enhancement is expected for spectra recorded with near-infrared (785 nm) excitation. Indeed, Fig. 5 demonstrates enhancement of predominantly Cu–SCys stretching vibrations coupled to Cys deformation motion at 367, 388, 409, 428, and 494 cm−1 [28]. In addition, a non-resonant Raman band from phenylalanine residues is visible as a shoulder at 1003 cm−1 . To probe DET of ThLc on AuNPs by SERS ThLc was adsorbed on 39 nm AuNP modified gold electrodes and SERS spectra were recorded at different electrode potentials in the absence of oxygen (Fig. 6). Pre-resonance enhancement is expected for spectra recorded with near-infrared (785 nm) excitation. However, T1 copper centre bands as noted in Fig. 5 are not present in SERS spectra shown by Fig. 6. The presence of the adsorbed protein at 0.8 V electrode potential is clearly evidenced from the sharp Phe ring stretching peak near 1004 cm−1 (F12 mode), a characteristic

Fig. 4. Enzyme turnover (kcat ) and DET (k0 ) constants obtained for TaLc (A) and ThLc (B) adsorbed on planar electrodes modified with AuNPs of different diameter. The constants have been calculated by processing data from simultaneous monitoring of (i) the mass of surface bound AuNPs and the mass of adsorbed enzyme by QCM-D and (ii) the current of oxygen bioelectroreduction at different applied potentials by linear sweep voltammetry. A Gaussian distribution model was fitted to the data points to resemble the bell-shaped dependence of rate constants obtained at electrodes modified with AuNPs of different diameter.

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Fig. 6. SERS spectra of ThLc, adsorbed on Au/AuNP electrode. SERS spectra of ThLc, adsorbed on an Au/AuNP electrode at 0.8 V (a), 0.6 V (b), 0.4 V (c), 0.2 V (d), 0.0 V (e), and 0.8 V (f) potentials (vs SHE) in the frequency regions 300–1800 cm−1 (A) and 2700–3100 cm−1 (B). The planar Au electrode for SERS measurements was modified with 39 nm AuNPs by drop coating. Experiments were performed in 50 mM Na2 HPO4 buffer solution (pH 4.0, adjusted with citric acid) containing 0.1 M Na2 SO4 . Measurement conditions: excitation wavelength – 785 nm; laser power at the sample – 30 mW; total integration time – 300 s.

Phe ring mode at 1031 cm−1 (F18 mode), and an aromatic ring ␯(=C − H) stretching vibration at 3059 cm−1 . The assignments of other vibrational modes of protein and adsorbed citrate ion are listed in Table 1. The gold electrode was not roughened for the purpose of enhancing Raman scattering. Thus, an observation of clear spectra from adsorbed protein indicates that AuNPs used for bioelectrocatalysis support the surface enhanced Raman scattering effect. The sharp 1004 cm−1 band broadens and shifts to lower wavenumbers (1001 cm−1 ) at more negative electrode potentials, while the high frequency component at 3059 cm−1 completely disappears at 0.0 V. Such downshift of the F12 mode frequency was found to be dependent both on the nature of the substrate and

Table 1 Tentative assignments of the SERS bands. Frequencya /cm−1

Assignment

Frequencya /cm−1

Assignment

729 824 868 1004 1031 1237 1304 1372

␯(C − S) Tyr (Y1) Trp (W17) Phe (F12) Phe (F18a) Amide III, ␤ sheet w(CH2 ), t(CH2 ) ␯s (COO) citrate

1440 1532 1595 1663 2950 2924 2965 3059

␦(CH2 ) ␯as (COO), ␦(NH2 ) ␯as (COO), ␦(NH2 ) Amide I ␯s (CH2 ) ␯(CH2 ) ␯as (CH3 ) ␯(=C − H) aromatic

a Determined at E = 0.80 V. Abbreviations: ␯, stretching; ␦, deformation; ␯s , symmetric stretching; ␯as , asymmetric stretching; t, twisting; w, wagging.

Table 2 Parameters of the Phe ring F12 and F18a vibrational modes of SERS spectra. Experimental conditions

Peak position/cm−1

Area/arb. un.

Intensity/arb. un.

Full width at half maximum/cm−1

0.8 V, before excursion to 0.0 V

1003.5 1031.0 1000.3 1028.2 1002.7 1030.4

543 668 617 665 471 597

76 44 62 45 64 41

6.0 10.4 9.3 12.1 6.7 11.2

0.0 V 0.8 V, after excursion to 0.0 V

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the electrode potential and was explained by direct interaction of Phe ring with the metal [29]. The reorientation of Phe rings to more parallel with respect to electrode surface orientation is in accordance with the observed decrease in ␯(=C − H) mode relative intensity [30]. Another argument for stronger interaction of adsorbed protein with electrode at less positive potentials (0.0 V) comes from the observation of “soft” C − H stretching mode (shoulder near 2825 cm−1 ) from the methylene groups of the amino acid side chains pointing on the direct interaction of CH2 groups with the surface [31]. SERS data suggest that at less positive electrode potential (0.0 V) the formation of a more extended flat configuration of surface bound protein takes place. This potentialdependent transformation is reversible as the parameters of the F12 band returned to near initial values (Table 2) and the “soft” mode disappeared when electrode potential was swept back to 0.8 V (Fig. 6). The SERS bands of the adsorbed protein are visible at all studied potentials indicating that the Lc is firmly bound to the AuNP surface. Co-adsorbed citrate anions on AuNPs are visible from the 1372 cm−1 band [32], and, probably, from the strong 1532 and broad 1595 cm−1 bands (at E = 0.8 V). The 1532 cm−1 band may have the contribution from the ␯as (COO) vibration from adsorbed decomposition products of citrate anion (acetonedicarboxylic acid and formate) [32–34]. The frequency of this band upshifts from 1532 to 1542 cm−1 by changing the electrode potential from 0.8 to 0.0 V, respectively (Fig. 6A). The observed spectral changes suggest the direct interaction of carboxylate group with the surface of AuNPs. Adsorption of citrate anion and its decomposition products may prevent denaturation of protein macromolecules at interface. SERS spectra provide evidence for almost irreversible adsorption of ThLc at Au/AuNP interface. Comparison of the areas of the Phe ring stretching modes (F12 and F18a) determined at 0.8 V before and after changing potential to 0.0 V (Table 2) showed that more than 86–89% of the enzyme remains adsorbed at Au/Au-NP surface during electrode polarisation, i.e. after approximately 1 h. Similar SERS experiments performed by using TaLc have not shown any significant differences from the SERS spectra obtained with ThLc. However, the intensity of the surface bound citrate band near 1375 cm−1 is higher at similar electrode potentials, while the protein bands are of lower intensity. The fact that the protein bands of TaLc in the SERS spectra have lower intensity probably indicates that TaLc is less conformationally changed on AuNPs if compared with ThLc. This suggestion is also in agreement with the higher kcat found for AuNP bound TaLc.

4. Conclusions In this work we found that TaLc and ThLc adsorb on planar Au electrode surfaces as well as on AuNPs. From combined ellipsometric and QCM-D measurements it was determined that adsorbed TaLc layer contains 68 ± 2 wt%, whereas the layer of ThLc contains 84 ± 5 wt% of water. The higher amount of water in the layer of surface bound ThLc could again be explained by a higher presumed degree of glycosylation (see above). TaLc has 27% lower mass, however, the degree of glycosylation is unknown. From SERS experiments it can be concluded that both laccases are almost irreversibly bound to AuNPs and that changing the applied potential induces some reversible reorientation of the enzyme molecules. Both TaLc and ThLc enzymes show efficient DET coupling with AuNPs although not with planar Au surface. It is important to mention that DET based bioelectrocatalytic oxygen reduction at TaLc modified AuNPs was demonstrated for the first time and was found to be superior to the bioelectrocatalysis obtained with the well-studied ThLc. By comparing the rate constants describing the bioelectrocatalytic process we found that the improvement of bioelectrocatalytic oxygen reduction is due to approximately

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