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Controlling the Surface Chemistry of Multiwalled Carbon Nanotubes for the Production of Highly Efficient and Stable Laccase-Based Biocatalysts [ChemPlusChem](ChemPlusChem Via Acquire Media NewsEdge) Laccase was immobilized over multiwalled carbon nanotubes (MWCNTs) with diameters ranging from 10 to 100 nm and different surface chemical groups. Oxygen-containing groups were introduced selectively or removed by liquid-phase oxidation and thermal treatments. The effect of pH on the immobilization efficiency and catalytic activity of laccase has been evaluated. Pristine MWCNTs show excellent immobilization capacity (100 %) and high enzyme activity, but low thermal stability (at 50 °C) owing to weak interaction (mostly hydrophobic) between laccase and the support. Immobilization capacity and catalytic activity of laccase on the modified MWCNTs were related to the surface electric charge of both the support and the enzyme. MWCNTs oxidized with HNO3 and posteriorly heated at 400 °C, mostly presenting hydroxyl surface groups, provided the best compromise between laccase activity and thermal stability, which has been attributed to the formation of hydrogen bonds between the support and the enzyme. Keywords : biocatalysis · enzymes · immobilization · nanotubes · surface chemistry (ProQuest: ... denotes formulae omitted.) Introduction The unique structure and properties (mechanical, optical, mag- netic, electronic, thermal, and biocompatibility) of carbon nanotubes (CNTs) have been studied intensively in different ad- vanced areas of application, such as electronics, energy stor- age, biotechnology, and environmental engineering.[1] Addi- tionally, CNTs offer the possibility to be functionalized, thus modifying their properties and improving their efficiency as supports or catalysts.[2] CNTs have been used in biocatalysis mainly as carriers for enzyme immobilization, particularly in biosensor development.[3-5] Compared to classical carriers, such as silica and polymeric materials,[6, 7] CNTs present some inter- esting advantages, such as large specific surface area, high ad- sorption capacity, high conductivity, and high enzyme loading capacity.[8, 9] In general, free enzymes are less stable and can be deactivated by extreme conditions of pH, temperature, and use of organic solvents. Immobilization of enzymes on solid carriers, and particularly on CNTs, is an interesting technique for improving their catalytic performance and stability, selectiv- ity, and reusability.[10] Different techniques for enzyme immobi- lization on CNTs have been described in the literature,[9] includ- ing : 1) direct physical adsorption ;[11] 2) adsorption onto CNTs functionalized with polymers,[12] biomolecules,[13] or surfac- tants ;[14] and 3) covalent bonding.[15] However, leaching of the enzyme from the carrier after a certain number of reutilization cycles has limited its application at laboratory and industrial scales. Moreover, it has been reported that in some cases the immobilization of enzymes on CNTs leads to a decrease in their catalytic activity.[3, 16, 17] Thus, the investigation of the struc- ture and function of enzymes immobilized on CNTs is funda- mental for understanding the interactions between the enzyme and the support, with the aim of developing very stable biocatalysts. Laccases (EC 1.10.3.2, p-diphenol oxidase) are attractive and industrially relevant enzymes belonging to the family of multi- copper oxidases.[18] Laccases have the capability of catalyzing the four-electron reduction of oxygen to water with concomi- tant oxidation of a broad range of substrates.[19] This enzyme can be used in various biocatalytic processes, such as pulp and paper delignification,[20] bioremediation including waste detoxi- fication,[21] and textile dye removal,[22, 23] and antimicrobial (in- cluding bactericidal and sporicidal) applications,[24] among others. Recently, some researchers have immobilized laccase on CNTs through adsorption or covalent bonding ;[4, 5, 25] howev- er, the relationship between the surface properties of this type of support and its immobilization capacity and thermal stabili- ty has not yet been explored. With the aim of developing highly efficient and robust bio- catalysts, we herein describe the effect of multiwalled carbon nanotube (MWCNT) surface functionalization, through oxida- tion and selective removal of oxygen-containing surface groups at different temperatures, on the immobilization effi- ciency, catalytic activity, and thermal stability of laccase. For comparison purposes, immobilization of laccase on the non- functionalized pristine material was also performed for MWCNTs with different diameter ranges. The free and immobi- lized enzyme was characterized by the catalytic behavior for 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonic acid) (ABTS) oxi- dation. Results and Discussion Immobilization of laccase on MWCNTs with different diame- ters A preliminary study on the influence of MWCNT diameter on the immobilization yield and activity of laccase was performed using MWCNTs with three classes of external diameter ranges : 10-20, 20-40, and 60-100 nm. It was observed that laccase was immobilized spontaneously on the different MWCNTs in the absence of any surface modification or coupling reagents. Globular structured laccase covering the sidewalls of MWCNTs could be observed by TEM analysis (Figure 1 a). In some cases the presence of enzyme in the interior of the tubes was also observed (Figure 1 b). The results presented in Table 1 show that the activity of im- mobilized laccase was influenced by the MWCNT diameter with the maximum activity obtained for the material with di- ameter within the range 10 to 20 nm. A linear relationship between the MWCNT average diameter and the immobilization yield and between this last parameter and laccase activity was found, as depicted in Figure 2. It has been reported that the increase in the curvature of CNTs could contribute to a decrease in the denaturing interac- tions between the enzyme and CNTs,[9] which may explain the higher activity of laccase immobilized on the MWCNTs with the lowest diameter range. Thus, the highly curved surfaces of CNTs are favorable for laccase activity and to suppress lateral interactions between adjacent adsorbed enzymes.[9] In the present case, the high surface area presented by the MWCNT with the lowest diameter (Table 1) may also play a role in the efficient loading of the enzyme along the MWCNT surface. So, the MWCNT with diameter range between 10 and 20 nm was selected for further studies. Effect of MWCNT surface chemistry on laccase immobiliza- tion and catalytic activity Direct physical adsorption is a commonly used noncovalent technique for immobilization of enzymes onto CNTs. The main driving forces involved in this process are associated with hy- drophobic, electrostatic, and p-p stacking interactions be- tween CNT sidewalls and enzymes[26] and also with hydrogen bonding.[8,26] These interactions depend on both enzyme side- chain functional groups and CNT surface properties. Laccase contains many ionizable groups on the side chains of the amino acids, such as amino and carboxyl groups.[27] The pH of the solution, and the pK of the side chain and its environment influence the charge on each side chain.[28] It has been report- ed that covalent immobilization of laccase on CNTs can be ach- ieved by reaction of free amine groups of terminal proteins with carboxylic acid groups generated at the surface of CNTs.[15] Nevertheless, this attachment generally occurs in the presence of mediators such as carbodiimide for carboxylic acid activation. Oxygenated functional groups can be formed spontaneously by exposure of carbon materials to the atmosphere. Neverthe- less, the concentration of these naturally formed groups is very low and can be further increased by oxidative treatments, in either the gas or liquid phase. Thermal treatments under nitro- gen atmosphere can then be used to remove selectively some of the groups formed.[29] The nature of these groups and their relative concentration dictates the acidity or basicity properties (from a Lewis standpoint) of the carbon material's surface. To study the interaction of laccase with the surface of MWCNTs and assess its importance on laccase immobilization yield and respective activity, MWCNT samples with different surface chemistry were prepared by liquid-phase oxidation and posterior thermal treatment of MWCNTs with diameter 10- 20 nm. The characterization of the prepared materials is sum- marized in Table 2. The surface areas of the samples, calculated by the BET method (SBET), revealed that the oxidation treatment led to an increase in the surface area of the resulting material. The sur- face area of MWCNTox is around 1.3 times higher than that of pristine MWCNT (Table 2). This occurs because oxidative treat- ment with HNO3 creates sidewall defects and can open up the end caps of the MWCNT, therefore increasing the porosity.[30] The oxidizing treatment produces materials with large num- bers of surface acidic groups, mainly carboxylic acids and, to a smaller extent, lactones, anhydrides, and phenol groups formed at the edges/ends and defects of graphitic sheets.[29, 30,32] The different surface oxygenated groups decom- pose by heating, thereby releasing CO and/or CO2, which were followed by temperature-programmed desorption (TPD) analy- sis (Figure 3). Oxygen surface groups on carbon materials are thermally decomposed at specific temperatures, thus making possible their identification from the TPD spectra.[31] The CO2 is released from the decomposition of carboxylic acid groups at low tem- peratures or from lactones at high temperatures, whereas phe- nols and carbonyl/quinone basic groups decompose as CO at high temperatures. Anhydrides are released as both CO and CO2 at intermediate temperatures.[31, 32] The total amounts of CO and CO2 evolved from the samples, obtained by integration of the TPD spectra, are presented in Table 2. The liquid-phase oxidation treatment with HNO3 produces a large number of acidic oxygen groups, which decompose to release CO2. Some of these groups, corresponding to carboxyl- ic acids, are removed by heating at 400 8C, phenols and car- bonyl/quinones remaining at the surface of the CNTs. Nearly total removal of surface groups is observed after treating MWCNTox at 900 8C. The MWCNTox sample presents the lowest ratio of CO/CO2, which indicates that this is the most acidic sample, whereas MWCNTox-900 presents the highest CO/CO2 ratio, thus sug- gesting the less acidic characteristics (Table 2). These observa- tions are consistent with the pHPZC results (Table 2). The acid character of the samples decreases on increasing the thermal treatment temperature, since the acid groups are removed at lower temperatures than neutral and basic groups, as reported in the literature.[31, 32] The ability of laccase to be immobilized onto the different functionalized MWCNT samples was investigated under differ- ent pH conditions (Figure 4 a). It can be observed that even without any further treatment, the pristine MWCNT shows ex- cellent (100 %) immobilization capacity over the range of pH values tested. In the case of the surface-modified materials, the relative immobilization yield varied with pH. This different be- havior between the original and the modified materials may foresee the existence of distinct interaction mechanisms be- tween the support and the enzyme. In the case of the original material, since no defects were created at the surface of the CNTs, hydrophobic and p-p stacking interactions are the most probable mechanisms of laccase immobilization. Laccase is a macromolecule that contains hydrophobic regions on its structure, which may adsorb on the external walls of CNTs through hydrophobic interactions.[26] The p-p stacking interac- tion between the sidewalls of pristine MWCNTs and the aro- matic rings present on the structure of laccase may also con- tribute to the adsorption of the enzyme.[33] Yet, for the modified materials, the differences in laccase im- mobilization performance can be mostly attributed to the exis- tence of electrostatic interactions between the support and the enzyme. Immobilization yields are in agreement with the charge properties of both CNTs and laccase, that is, the pHPZC of the carbon materials and the isoelectric point of laccase (pI = 4.2).[34] Among the modified materials, MWCNTox-400 presents the lowest immobilization efficiency over the pH range tested, which may be related to the proximity between the pHPZC of the carbon material (4.0) and the pI of the enzyme. For the material heat-treated at 900 8C, more intense electrostatic interactions are expected for pH values between 4.2 and 6.9, conditions at which the carbon support is positive- ly charged and the enzyme is negatively charged. On the other hand, MWCNTox, which has a pHPZC of 3.0, presents the highest immobilization capacity, being similar to that of pristine MWCNTs for pH ^ 4. In the pH range 3-4, the MWCNTox surface is negatively charged whereas laccase is positively charged, thus leading to the existence of a strong electrostatic interaction between both the support and the enzyme. Moreover, the immobilization yield and activity (Fig- ure 4 b) of the enzyme immobilized on the MWCNTox support were higher than those observed for the other modified mate- rials over the total pH range tested, which may indicate that stronger interactions besides pure electrostatic attraction may play a role. To further investigate CNT-laccase interactions, FTIR-attenu- ated total reflectance (ATR) spectra of MWCNTox and MWCNTox-400 before and after laccase immobilization were re- corded (Figure 5). In the case of MWCNTox-900, the infrared signal could hardly be measured since most of the oxygen sur- face groups are removed upon thermal treatment of the oxi- dized support at 900 8C. For the MWCNTox material (Figure 5 a), four main bands could be identified,[35] which confirmed the presence of the oxygenated surface groups : 1) a band at 1800 cm^1 attributed to the C=O stretching vibration in lac- tones, carboxylic acids, and anhydrides ;[33] 2) a broad band as- signed to O^H bending in phenols and carbonyls centered at 1400 cm^1; 3) a band at 1020 cm^1 assigned to the C^O stretching vibration in ethers and phenols ; and 4) a band at 880 cm^1 ascribed to isolated aromatic C^H out-of-plane bend- ing mode vibration. In the case of MWCNTox-400 (Figure 5 b), the intensity of the band at 1800 cm^1 decreased significantly owing to the removal of carboxylic acid groups after the ther- mal treatment at 400 8C, as previously determined by TPD anal- ysis. Moreover, the band at 1020 cm^1 became more intense, probably as a result of the decrease in the electronic and spa- tial interactions between ether and phenol groups with car- boxylic acid groups. Finally, the aromatic out-of-plane C^H bending band appears deviated peaking at 785 cm^1. After laccase immobilization on MWCNTox and MWCNTox- 400, some changes in the infrared spectra were observed. When MWCNTox was used as support, an expressive decrease in the intensity of the bands attributed to O^H bending and C=O stretching was detected (Figure 5 a). In addition, the ap- pearance of two new bands at 1650 and 1320 cm^1, assigned to N^H bending and C^N stretching vibration of amines, re- spectively, has been recorded. These results suggest that, in this case, physical adsorption of the enzyme on MWCNTox on carboxylic acid sites is the main interaction mechanism. In the case of laccase immobilized on MWCNTox-400 (Figure 5 b), an increase in the intensity of the C=O stretching band occurred, which can be attributed to exposed carboxylic acid terminal groups of laccase.[27] Moreover, the N^H bending band is slightly deviated, peaking at 1605 cm^1. This behavior reveals the existence of different interactions between this carrier and laccase, namely the formation of hydrogen bonds between OH groups at the surface of the nanotubes and amine terminal groups of the enzyme, as already reported for other supports such as zeolites and spent grain.[36, 37] Thermal stability of free and immobilized laccase Thermal stability experiments were performed with free and immobilized laccase. The loss in enzyme activity on incubation in buffer solution at 50 8C was taken as a measure of thermal stability. From the experimental data, kinetic thermal parame- ters were obtained to evaluate the thermal stability of the enzyme. The residual activity of free and immobilized laccase was monitored at regular intervals between 0 and 3.5 hours. Thermal deactivation parameters (a and k) and half-life time (t1= ) for free and immobilized laccase are reported in Table 3. Results show that the residual thermal stability of the immobi- lized laccase was improved in comparison to the free enzyme. Although initial inactivation is faster for immobilized laccase (resulting in a higher k parameter, Table 3), the remaining activ- ity increases with the immobilization along the period of time studied. The a parameter (remaining enzyme activity) was su- perior for immobilized laccase within all supports tested, at- taining a maximum of 74.2 (from 0 to 100) for MWNTox-400. This result corresponds to an increase of 2.3 times in compari- son to free enzyme. As discussed previously, the availability of OH groups in MWCNTox-400 can promote a more stable at- tachment between the enzyme and support, probably by for- mation of hydrogen bonds between them.[37] Thus, the in- creased residual stability observed for the immobilized laccase on MWCNTox-400 may be attributed to a decrease in the enzyme structure mobility, thereby avoiding the denaturing ef- fects of the environment. According to the half-life time, the free enzyme lost 50 % of its initial activity after 14.4 hours of incubation at 50 8C (not shown), whereas for the immobilized laccase, t1= occurs at 2.8 and 2.1 hours when MWCNT and MWCNTox-900 are used as supports, respectively. On the other hand, the activity of lac- case immobilized on MWCNTox and MWCNTox-400 remained above 50 % of its initial value and therefore no half-life time parameters could be determined. The above results show that the surface functionalization of CNTs with oxygen-containing groups plays an important role in improving the residual thermal stability of laccase. MWCNTox and MWCNTox-400, which contain mostly carboxylic acid and phenol surface groups, respectively, were the sup- ports that provided a more stable laccase immobilization. On the other hand, when laccase was immobilized on MWCNT and MWCNTox-900, which have negligible amounts of surface groups, some loss of activity is observed with time owing to the weaker interactions between these supports and the enzyme. Conclusion Laccase was immobilized efficiently on multiwalled carbon nanotubes (MWCNTs) by a simple contact method. The diame- ter of the carbon nanotubes (CNTs) as well as their surface functionalization with oxygen-containing groups were found to be determinant for the efficient immobilization and catalytic activity of laccase-CNT hybrids. Laccase immobilized on CNTs with smaller diameter shows higher activity owing to a decrease in the denaturing interac- tions between the enzyme and the support promoted by the increase of the curvature of the nanotubes. Immobilization efficiency and enzyme activity appear to be related to the surface properties of the CNTs. For the pristine material, the main driving forces are hydrophobic interactions between the support and the enzyme. For the functionalized materials, electrostatic interactions and hydrogen bonding are the main mechanisms involved in the immobilization process. Among the modified materials, MWCNTox showed the high- est laccase immobilization capacity and catalytic activity, this behavior being attributed to a strong electrostatic interaction between the support and the enzyme promoted by the pres- ence of surface carboxylic acid groups. MWCNTox-400, the sur- face of which is mainly characterized by the presence of hy- droxyl groups, showed improved thermal stability, which is at- tributed to the stabilization of the enzyme following hydrogen bonding through the surface OH groups of the support. Experimental Section Materials and chemicals Pure commercial laccase (Novozym^ 51003; 1000 LAMU g^1) from genetically modified Aspergillus oryzae was kindly provided by No- vozymes, Denmark. MWCNTs with different external diameter ranges (10-20, 20-40, or 60-100 nm), synthesized by chemical vapor deposition, were purchased from Shenzhen Nanotechnolo- gies Co. Ltd. (purity ^95%, length = 5-15 mm, ash content ^ 0.2 wt %, amorphous carbon < 3 %). 2,2 ' -Azino-bis(3-ethylbenza- thiazoline-6-sulfonic acid) (ABTS) was supplied by AppliChem (Ger- many). Functionalization and characterization of MWCNTs MWCNTs with a diameter range of 10-20 nm were functionalized as follows. Oxidation byheating at reflux withHNO3 (7m)for3h at 130 8C was performed, followed by washing with distilled water until neutral pH, and drying overnight at 110 8C (sample MWCNTox). The MWCNTox material was heat treated under an inert atmosphere (N2) at 400 8C for 1 h (sample MWCNTox-400) and at 900 8C for 1 h (sample MWCNTox-900), to selectively remove sur- face groups. The obtained samples were characterized by N2 ad- sorption-desorption isotherms at ^196 8C. Brunauner-Emmett- Teller (BET) specific surface areas (SBET) were calculated from nitro- gen adsorption in the relative pressure range from 0.05 to 0.15. The Barrett-Joyner-Halenda (BJH) method was applied to the de- sorption branch of the N2 adsorption isotherms to obtain the pore size distribution curves and cumulative volume of pores. Surface chemical groups were characterized and quantified by tempera- ture-programmed desorption (TPD). The total amounts of CO and CO2 evolved from the samples were obtained by integration of the TPD spectra. The pH of the point of zero charge (pHPZC) of the MWCNT samples was determined by using a drift method de- scribed elsewhere.[38] Fourier transform infrared (FTIR) measure- ments were performed on a FTIR Nicolet 510-P spectrometer (Thermo Fisher Scientific, USA) equipped with a MIRacle^ Single Reflection ATR (attenuated total reflectance ZnSe crystal plate) ac- cessory (PIKE Technologies, USA). A JEOL 2010F analytical electron microscope equipped with a field-emission gun was used for trans- mission electron microscopy (TEM) investigations. Immobilization technique In all experiments 4 mg of MWCNTs were added to 1.2 mL of lac- case solution (3.75 mL laccase per mL buffer solution) under orbital stirring for 1 h. After immobilization, the MWCNTs were washed several times with an appropriate buffer. To evaluate the influence of pH on laccase immobilization, different buffers were used : 50 mm citrate/phosphate buffer for pH values of 3.0, 4.0, and 5.0 ; 50 mm phosphate buffer for pH 6.0, 7.0, and 8.0 ; and 50 mm car- bonate buffer for pH 9.0. Immobilization yield (%) is defined as the difference between the initial free enzyme activity of the laccase solution before the immobilization and the activity of free laccase obtained in the supernatant after immobilization divided by the enzyme activity before immobilization. Measurements of activity of free and immobilized laccase The free laccase activity was assayed spectrophotometrically (JASCO V-560 UV/Vis spectrophotometer) with ABTS as substrate (0.4 mm)in50mm citrate/10 mm phosphate buffer at pH 4.5. To measure the laccase activity, the incubated enzyme solution (0.1 mL) was added to the ABTS solution (1.9 mL) at 40 8C.[39] The change in absorbance at 420 nm (e =36000m^1cm^1) was record- ed automatically by the spectrophotometer and the catalytic activi- ty was determined by measuring the slope of the initial linear por- tion of the kinetic curve. One unit (U) was defined as the amount of enzyme that oxidized 1 mmol of ABTS per minute and the activi- ties were expressed in U L^1. To measure laccase activity when using immobilized enzyme, MWCNTs were mixed with citrate/phos- phate buffer (50/10 mm, 105 mL, pH 4.5) at 40 8C and ABTS (0.4 mm, 37.5 mL) under magnetic stirring at 100 rpm for 4 min. Samples were taken every minute, filtered with 0.45 mm polypropy- lene filters, and the absorbance was measured spectrophotometri- cally at 420 nm. After linear regression of the data obtained, enzyme activity was determined by using Equation (1): ... (1) in which U/g is the quantity of enzyme able to oxidize 1 mmol of ABTS per minute and per mass unit of carrier ; abs/min is the ab- sorbance per minute determined by linear regression ; fdil is the di- lution factor of the sample ; Vrxn (L) is the volume of reaction ; 106 is the conversion factor from m to mm ; e is the ABTS molar absorp- tion coefficient (36000m^1 cm^1 at 420 nm); and mCNT (g) is the mass of the MWCNT. Thermal stability of free and immobilized laccase The thermal stabilities of the free and immobilized laccase were in- vestigated by incubating the free and immobilized enzymes in car- bonate buffer (100 mm, pH 9.0) at 50 8C. Immobilized laccase was suspended in 3 mL of the buffer. The sample was removed regular- ly from the water bath and the enzymatic activity was determined quickly according to the methods described above. The kinetic thermal parameters were calculated according to the simplified de- activation model proposed by Arroyo et al.[40] [Eqs. (2) and (3)]: ... (2) ... (3) in which A is the residual enzyme activity, a the ratio of specific ac- tivities (remaining activities) E1/E, k the first-order rate constant of thermal inactivation, and t the time. The thermal parameters a and k of the model were estimated by a nonlinear fitting of the experi- mental data using CurveExpert v1.3 software. Biocatalyst half-life (t1= ) was calculated from Equation (3) by using the estimated pa- rameters (k and a) and making A equal to 50. Acknowledgements This work was supported by Project PEst-C/EQB/LA0020/2013, fi- nanced by FEDER through COMPETE-Programa Operacional Factores de Competitividade, and by FCT-Fundażo para a CiÞn- cia e a Tecnologia, and co-financed by QREN, ON2, and FEDER (Project NORTE-07-0124-FEDER-0000015). C.G.S. thanks FCT for the Postdoctoral Fellowship (SFRH/BPD/48777/2008). A.P.M.T. ac- knowledges the financial support (Programme CiÞncia 2008) from FCT. A.M.T.S. and G.D. acknowledge the financial support of the Slovenian Research Agency. A.M.T.S. acknowledges the FCT In- vestigator 2013 Programme (IF/01501/2013), with financing from the European Social Fund and the Human Potential Operational Programme. [1] X.-L. Xie, Y.-W. Mai, X.-P. Zhou, Mater. Sci. Eng. 2005, 49, 89 - 112. [2] V. Mittal, Carbon Nanotubes Surface Modifications : An Overview, Wiley- VCH, Weinheim, Germany, 2011. [3] D. Das, P. K. Das, Langmuir 2009, 25, 4421 - 4428. [4] R. P. Ramasamy, H. R. Luckarift, D. M. Ivnitski, P. B. Atanassov, G. R. John- son, Chem. Commun. (Camb). 2010, 46, 6045 - 6047. [5] H. L. Pang, J. Liu, D. Hu, X. H. Zhang, J. H. Chen, Electrochim. Acta 2010, 55, 6611 - 6616. [6] A. Salis, M. Pisano, M. Monduzzi, V. Solinas, E. Sanjust, J. Mol. Catal. B 2009, 58, 175 - 180. [7] C. Silva, C. J. Silva, A. Zille, G. M. Guebitz, A. Cavaco-Paulo, Enzyme Microb. Technol. 2007, 41, 867 -875. [8] W. Feng, P. Ji, Biotechnol. Adv. 2011, 29, 889 - 895. [9] N. Saifuddin, A. Z. Raziah, A. R. Junizah, J. Chem. 2013 , 2013, 676815. [10] C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan, R. Fernan- dez-Lafuente, Enzyme Microb. Technol. 2007, 40, 1451 - 1463. [11] J. T. Cang-Rong, G. Pastorin, Nanotechnology 2009, 20 , 255102. [12] X. Wu, B. Zhao, P. Wu, H. Zhang, C. Cai, J. Phys. Chem. B 2009, 113, 13365-13373. [13] G. D. Withey, J. H. Kim, J. Xu, Bioelectrochemistry 2008, 74, 111-117. [14] R. J. Chen, S. Bangsaruntip, K. A. Drouvalakis, N. Wong Shi Kam, M. Shim, Y. Li, W. Kim, P. J. Utz, H. Dai, Proc. Natl. Acad. Sci. USA 2003, 100, 4984-4989. [15] P. Asuri, S. S. Karajanagi, E. Sellitto, D.-Y. Kim, R. S. Kane, J. S. Dordick, Biotechnol. Bioeng. 2006, 95, 804 - 811. [16] S. S. Karajanagi, A. A. Vertegel, R. S. Kane, J. S. Dordick, Langmuir 2004, 20, 11594 - 11599. [17] P. Asuri, S. S. Bale, R. C. Pangule, D. A. Shah, R. S. Kane, J. S. Dordick, Langmuir 2007, 23, 12318- 12321. [18] M. Alcalde in Laccases : Biological Functions, Molecular Structure and In- dustrial Applications, Springer, Netherlands, 2007, pp. 461 - 476. [19] P. J. Strong, H. Claus, Crit. Rev. Environ. Sci. Technol. 2011, 41, 373 - 434. [20] A. P. M. Tavares, J. A. F. Gamelas, A. R. Gaspar, D. V. Evtuguin, A. M. R. B. Xavier, Catal. Commun. 2004, 5, 485 - 489. [21] A. Steevensz, M. M. Al-Ansari, K. E. Taylor, J. K. Bewtra, N. Biswas, J. Chem. Technol. Biotechnol. 2009, 84, 761 - 769. [22] R. O. Crist^v¼o, A. P. Tavares, L. A. Ferreira, J. M. Loureiro, R. A. Boaven- tura, E. A. Macedo, Bioresour. Technol. 2009, 100, 1094- 1099. [23] R. O. Crist^v¼o, A. P. Tavares, J. M. Loureiro, R. A. Boaventura, E. A. Macedo, Bioresour. Technol. 2009, 100, 6236 - 6242. [24] N. Grover, I. V. Borkar, C. Z. Dinu, R. S. Kane, J. S. Dordick, Enzyme Microb. Technol. 2012, 50, 271 - 279. [25] Y. Li, X. Huang, Y. Qu, J. Chem. Technol. Biotechnol. 2013, 88, 2227 - 2232. [26] Y. Gao, I. Kyratzis, Bioconjugate Chem. 2008, 19 , 1945 - 1950. [27] K. Piontek, M. Antorini, T. Choinowski, J. Biol. Chem. 2002 , 277, 37663 - 37669. [28] S. Kim, A. Cavaco-Paulo, Appl. Microbiol. Biotechnol. 2012, 93 , 585 - 600. [29] J. L. Figueiredo, M. F. R. Pereira, Catal. Today 2010, 150 ,2-7. [30] M. Monthioux, B. W. Smith, B. Burteaux, A. Claye, J. E. Fischer, D. E. Luzzi, Carbon 2001, 39, 1251 - 1272. [31] J. L. Figueiredo, M. F. R. Pereira, M. M. A. Freitas, J. J. M. ^rf¼o, Carbon 1999, 37, 1379 - 1389. [32] J. L. Figueiredo, M. F. R. Pereira, M. M. A. Freitas, J. J. M. ^rf¼o, Ind. Eng. Chem. Res. 2007, 46, 4110 - 4115. [33] K. Matsuura, T. Saito, T. Okazaki, S. Ohshima, M. Yumura, S. Iijima, Chem. Phys. Lett. 2006, 429, 497 - 502. [34] J. Kulys, R. Vidziunaite, P. Schneider, Enzyme Microb. Technol. 2003, 32, 455-463. [35] J. Chen, Q. Chen, Q. Ma, J. Colloid Interface Sci. 2012, 370, 32 - 38. [36] A. M. da Silva, A. P. M. Tavares, C. M. R. Rocha, R. O. Crist^v¼o, J. A. Teix- eira, E. A. Macedo, Process Biochem. 2012, 47, 1095 - 1101. [37] G.-W. Xing, X.-W. Li, G.-L. Tian, Y.-H. Ye, Tetrahedron 2000, 56, 3517 - 3522. [38] J. Rivera-Utrilla, I. Bautista-Toledo, M. A. Ferro-Garca, C. Moreno-Castilla, J. Chem. Technol. Biotechnol. 2001, 76, 1209 - 1215. [39] P. Ander, K. Messner, Biotechnol. Tech. 1998, 12, 191 - 195. [40] M. Arroyo, J. M. a. S^nchez-Montero, J. V. Sinisterra, Enzyme Microb. Technol. 1999, 24, 3- 12. Received : March 8, 2014 Published online on May 20, 2014 Cl^udia G. Silva,*[a] Ana P. M. Tavares,[a,b] Goran Drazic',[c,d] Adri^n M. T. Silva,[a] Jos^ M. Loureiro,[b] and Joaquim L. Faria[a] [a] Dr. C. G. Silva, Dr. A. P. M. Tavares, Dr. A. M. T. Silva, Prof. J. L. Faria LCM-Laboratory of Catalysis and Materials Associate Laboratory LSRE-LCM, Faculdade de Engenharia Universidade do Porto Rua Dr. Roberto Frias s/n, 4200-465 Porto (Portugal) E-mail : [email protected] [b] Dr. A. P. M. Tavares, Prof. J. M. Loureiro LSRE-Laboratory of Separation and Reaction Engineering Associate Laboratory LSRE-LCM, Faculdade de Engenharia Universidade do Porto Rua Dr. Roberto Frias s/n, 4200-465 Porto (Portugal) [c] Prof. G. Drazic' Department of Nanostructured Materials Jozef Stefan Institute Jamova 39, 1000 Ljubljana (Slovenia) [d] Prof. G. Drazic' Laboratory for Materials Chemistry National Institute of Chemistry Hajdrihova 19, 1000 Ljubljana (Slovenia) (c) 2014 Blackwell Publishing Ltd. |