Immobilization of peroxidase on functionalized carbon nanotubes for synthesis of biocatalysts with high performance

Master of Sciences in Bioengineering Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Master’s Thesis by Renato Alexandre Moreira Azevedo Dissertation for Master degree in Biological Engineering performed in Associate Laboratory LSRE-LCM, Department of Chemical Engineering Faculdade de Engenharia, Universidade do Porto Supervisors: Drª. Ana Paula Tavares and Drª. Cláudia Gomes Silva July, 2014 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Declaration It is declared on oath that this work is original and that all non-original contributions have been properly referenced with the identification of the source. Renato Alexandre Moreira Azevedo DEPARTMENT OF CHEMICAL ENGINEERING Tel. +351-22-508 1884 Fax +351-22 508 1449 Published by FACULTY OF ENGINEERING OF THE UNIVERSITY OF PORTO Rua Dr. Roberto Frias 4200-465 PORTO Portugal Tel. +351-22-508 1400 Fax +351-22-508 1440 [email protected] http://www.fe.up.pt Author’s information: Renato Alexandre Moreira Azevedo 200905207 bio0908[email protected] Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance This page intentionally left empty Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance “The only source of knowledge is experience.” Albert Einstein Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance This page intentionally left empty Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Acknowledgements A project results invariably of a conjoined set of efforts. The present work fits this rule. Although this is a document for which I assume total responsibility, the reality is that its merit is not entirely mine. I, therefore, dedicate this first segment to those that, in a way or another, have fostered my academic growth and have guided me in this master thesis. Firstly, the continuous guidance of my supervisors, Drª. Ana Paula Tavares and Drª. Cláudia Gomes Silva must be highlighted. Their knowledge, experience and motivation made this work possible. Evidently, my close relatives, friends and girlfriend were, likewise, an import pillar. I could always count with their unconditional support, without which this project couldn’t be done. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance iv Fig. A5 - XRD diffractogram of magnetite (Fe3O4) and CNT-Fe3O4 hybrid materials. ....................... 83 Fig. A6 – mMWCNT attracted by a simple magnet ................................................................. 84 Fig. A7 - TEM images of Fe3O4 (a) and mMWCNTs (b, c and d). ................................................. 85 Fig. A8 – Arrhenius plot for free and immobilized peroxidase on MWCNTox-400. .......................... 87 Fig. A9 - Thermal deactivation of free peroxidase and immobilized on purified and functionalized MWCNT at 40 ºC ......................................................................................................... 88 Fig. A10 - Thermal deactivation of free and immobilized peroxidase on purified and functionalized MWCNT at 50 ºC ......................................................................................................... 89 Fig. A11 - Effect of H2O2 concentration on the initial rate of oxidation of ABTS (0.4 mM) catalyzed by free peroxidase .......................................................................................................... 90 Fig. A12 – Lineweaver-Burk representation of free peroxidase ................................................. 91 Fig. A13 - Lineweaver-Burk representation of immobilized peroxidase ...................................... 91 Fig. A14 – Reusability of peroxidase immobilized on MWCNTox-400 .......................................... 93 Fig. A15 – Reusability of peroxidase immobilized on a MWCNTs membrane ................................. 93 Fig. A16 – Standard curves for Bradford protein assay ........................................................... 98 Fig. A17 – Isoelectric point focusing. Lac (Laccase), Stdr. (Standard) and Perox (Peroxidase), pH 4-6. ............................................................................................................................. 100 Fig. A18 – SDS-Page image for peroxidase (~31 kDa) and for laccase (~80 kDa) ............................ 103 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance v Tables List Table 1 – A summary of the major CNTs production methods and their efficiency ...........................6 Table 2 – Application of enzymes immobilized on carbon nanotubes.......................................... 11 Table 3 - Technological properties of immobilized enzyme systems .......................................... 13 Table 4 - Classification of supports .................................................................................. 14 Table 5 - Selected characteristic parameters of immobilized enzymes ....................................... 15 Table 6 – Molecular properties of a fungal peroxidase ARP ..................................................... 20 Table 7 - Specific surface area , pore volume and pore diameter for the different MWCNT samples. ................................................................................................................... 35 Table 8 – Peroxidase immobilization yield on the MWCNT materials for the different pH ............... 44 Table 9 - Recovered activity of the immobilization process for the different pH of the medium used to perform immobilization ................................................................................................ 44 Table 10 - Thermal parameters and thermal stabilities obtained for the thermal inactivation of free and immobilized peroxidase ........................................................................................... 48 Table 11 – Thermal parameters (50 ºC) for free and immobilized peroxidase on purified and functionalized MWCNT ................................................................................................. 50 Table 12 – Kinetic parameters of ABTS oxidation for free and immobilized peroxidase on purified MWCNTs ................................................................................................................... 53 Table 13 – Dye discoloration % of RB5, RB and BR by free and immobilized peroxidase on mMWCNTs . 56 Appendix Table A1 - Desorption temperature of oxygen functional groups in carbon materials ..................... 79 Table A2 - Characterization of Fe3O4 and CNT-Fe3O4 hybrid material. ........................................ 83 Table A3 - Summary table for test at pH range 4.5-9 ............................................................ 86 Table A4 - Thermal parameters (40 ºC) for free and immobilized peroxidase on purified and functionalized MWCNT ................................................................................................. 88 Table A5 – Comparison of Michaelis-Menten parameters obtained for free peroxidase ................... 91 Table A6 - Comparison of Michaelis-Menten parameters obtained for immobilized peroxidase ......... 92 Table A7 – Properties of Reactive Black 5 .......................................................................... 94 Table A8 - Properties of Reactive Blue 4............................................................................ 95 Table A9 - Properties of Bromophenol Blue ........................................................................ 96 Table A10 – Protein quantification for the immobilization process ........................................... 99 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance vi Glossary Abbreviations ABTS 2,2’-azino-bis(3-ethylbenzathiazoline-6-sulfonic) acid AMG Amyloglucosidase APTS (3-Aminopropyl)triethoxysilane APX Pea ascorbate peroxidase Arg Arginine ARP Arthromyces ramosus peroxidase BAP Lignin peroxidase from Bjekandera adusta BCA Bicinchoninic acid BET Brunauner-Emmett-Teller BJH Barrett-Joyner-Halenda BR Bromophenol Blue CcP Cytochrome-c peroxidase CD Circular dichroism spectroscopy CFX Ciprofloxacin CNT Carbon nanotube Cys Cysteine EDC N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride ELISA Enzyme-liked immunosorbent assay FTIR Fourier transform infrared His Histidine HRP Horseradish peroxidase LiP Lignin peroxidase MnP Manganese peroxidase MNP Magnetite nanoparticle MWCNT Multi-walled carbon nanotube NHS N-hydroxysuccinimide PCP Lignin peroxidase of P. chrysosporium pI Isoelectric point RB Reactive blue 4 RB5 Reactive black 5 Rz Reinheitszahl value SBP Soybean peroxidase SDS Sodium dodecyl sulfate Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance vii SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Scanning electron microscope SWCNT Single-walled carbon nanotube TEM Transmission electron microscopy TH Thionine THF Tetrahydrofuran TPD Temperature programmed desorption UV Ultraviolet XRD X-ray diffraction Substrate Variables Pore diameter nm Michaelis-Menten constant mM Specific surface area m2.g-1 Pore volume cm3.g-1 pH value at the point of zero charge Maximum rate of the reaction mM.min-1 Abs Absorbance m2 Ea Arrhenius activation energy kJ.mol-1 h Planck constant J.h k Thermal inactivation rate constant h-1 K Boltzmann constant J.K-1 t1/2 Half-life time h α Ratio of specific activitiy ε Molar extinction coefficient M-1.cm-1 Catalytic reaction rate mM.min-1 Standard free energy for the thermal inactivation J.mol-1 Activation enthalpy kJ.mol-1 Activation entropy kJ.mol-1 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance viii Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Introduction 1 1 Introduction 1.1 Background and Project Presentation Nanotechnology is the ability to understand, create, and use material structures devices and systems with fundamentally new properties and functions at the atomic, molecular, and supramolecular levels (Drexler, 1996). In addition to physical and chemical sciences, one of the most interesting objectives of nanotechnology might be described by the ability to assemble biological molecules into nanostructured materials, i.e., the relationship with biology field. The ability to understand the structure and function of biological systems at the nanoscale, along with the controlled production (geometries, dimensions, surface properties) of nanomaterials, has led to a new field: nanobiotechnology. It is defined as the application of the tools and techniques developed by nanotechnology to understand and transform biosystems and which uses biological principles and materials to create new functional nanostructures (Roco, 2003). Thus, there is now an increasing interest in understanding and controlling the interactions of nanomaterials with biological molecules, such as proteins With unique chemical, electronic, mechanical and biocompatibility properties (Balasubramanian, 2006), carbon nanotubes (CNTs) have been extensively investigated for various applications during the last decade, especially in biomedical imaging, drug delivery, biosensing, biocatalyst and in the design of functional nanocomposites. These robust nanoscaffolds have an inherently large surface area, which leads to high enzyme loading and consequently high enzyme activity (Silva et al., 2014). In general, free enzymes are less stable and prone to denaturation in extreme conditions of pH, temperature and organic solvents. To improve their stability, enzymes have generally been immobilized on a solid carrier, which is expected to enhance catalytic stability, selectivity and reusability of the enzymes. The most used strategies for enzyme immobilization on CNTs described in the literature include adsorption and covalent bonding. Adsorption is a relatively simple method as it is a chemical free enzyme binding process. However, leaching of the enzyme from the immobilized enzyme preparation after a certain number of reuses has limited its application at a commercial scale. On the other hand, covalent binding methods produce relatively stable immobilized enzyme preparations with more reusability as compared to the physical adsorption method. Therefore, the investigation on the structure and function of enzymes immobilized on CNTs is fundamental in order to understand the interactions between the enzyme and the support, with the aim at developing very stable biocatalysts. Additionally, CNTs offer the possibility of being functionalized, thus modifying their properties and improving their efficiency as supports or catalysts. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Introduction 2 The surface functionalization of nanomaterials involves grafting of desirable functional groups onto their surface to obtain nanomaterials with desired properties (Shim et al., 2002). This functionalization can change their dispersibility and interactions with enzymes and consequently affect the catalytic activity of the immobilized enzyme. It has been demonstrated that biological and bioactive species such as proteins, carbohydrates and nucleic acids can be conjugated with carbon nanotubes, either by covalent and noncovalent conjugations (Thordarson et al., 2006; Wei et al., 2007). It is important to study the behavior of the immobilized enzyme under different conditions, such as pH, temperature and enzyme concentration. Investigating the structure and function of enzymes immobilized on nanomaterials will be crucial for developing a better understanding of enzyme-nanomaterial interactions, as well as for designing functional protein-nanomaterial conjugates. Peroxidases are attractive and industrially relevant enzymes that belong to the group of oxidoreductases which catalyse the reduction of peroxides to form water and the oxidation of a variety of organic and inorganic compounds (Johannes Everse et al., 1990). Peroxidase has been immobilized on CNTs through adsorption or covalent bonding for various applications, such as biolectrochemical sensor, fuel cells, glucose and H2O2 biosensor and, as in this work, the use in biocatalytsts. However, information about the relationship between surface properties of this type of supports and its immobilization capacity and thermal stability have been to the date scarcely explored. 1.2 Main Objectives All biological systems have the first level of organization at the nanoscale where their fundamental properties and functions are defined. A variety of enzymes, including peroxidases, have been attached to a suite of nanomaterials, in particular carbon nanotubes, for various applications, such as sensing, drug delivery and biocatalysis. However, very few studies have elucidated the influence of the nanoscale environment on structure and function of this enzyme. This present work describes the effect of MWCNTs surface functionalization through oxidation and selective removal of oxygen-containing surface groups at different temperatures, on the immobilization efficiency, catalytic activity and thermal stability of peroxidase, with the main goal to design optimal MWCNT-peroxidase conjugates for applications relevant to biocatalysis. Therefore, the objectives of this work are: - To produce and to characterize functionalized MWCNTs through selectively introduction or removing of oxygen containing groups. - To evaluate the structure, function, and stability of peroxidase on different MWCNTs with the overall aim of synthesis of biocatalysts with high performance. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Introduction 3 - To study the biocatalytic capacity of free and immobilized peroxidase for degradation of textile dyes. 1.3 Thesis organization This thesis work is divided into 7 chapters, with the last two being the chapter of references and appendix, respectively. Chapter 1 describes the main goals of this work, background and motivations for its developments. It serves as a guideline to the overall work presented in the further chapters. In Chapter 2 a brief review of the literature is provided, situating the work within the context of existing published reports. This chapter is divided into three parts: in the first one, a theoretical introduction to carbon nanotubes is made, referring its main features and advantages as well as production methods. The different functionalization techniques are also discussed, ending with the various applications of enzyme immobilization on this material. Continuing, in the second part, a general overview of enzyme immobilization is made. Their advantages and disadvantages and immobilization techniques are presented as well as the importance of the enzyme-carrier relationship. The last part is related to the enzyme used in this work peroxidase Information about its structure and composition as well as its properties and potential applications are described. Finally, a revision of literature on immobilization of peroxidase on carbon nanotubes is presented. Chapter 3 refers to materials and methods of experimental work of this thesis. The first part focuses on the production and characterization of the CNTs used. The second part presents the methodology used for the immobilization of peroxidase and for all tests associated with MWCNT-peroxidase conjugates. At the end, this complex was applied to degradation of dyes. In chapter 4, a presentation of results with discussion is made, following the same pattern of the previous chapter. Firstly, the changes on the surface of carbon nanotubes caused by oxidation and thermal treatment were discussed, as well as the possibility of CNT magnetization. The effect of functionalization on the immobilization of peroxidase was evaluated by comparing the results of activity and immobilization yield. After analyzing the optimal immobilization conditions, the stability and kinetic constants of the immobilized enzyme with the enzyme in its free state is compared. Finally, the discoloration yield of 3 textile dyes was assesed using free and immobilized peroxidase. Finally, chapter 5 gives an overview of the work presented, describing the main conclusions and showing proposals for future research with the possible limitations or possibilities for further studies. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Introduction 4 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 5 2 Literature Review 2.1 Carbon Nanotubes Among the huge amount of nanomaterials, carbon nanotubes (CNTs), discovered in 1991 by Iijima (Iijima, 1991) have been extensively investigated for various applications during the last decade owing to their unique chemical, electronic and mechanical properties (Balasubramanian and Burghard, 2005; Gong K.P., 2005; Trojanowicz, 2006). CNTs consist of graphitic sheets rolled up into a cylindrical shape with lengths in the micrometers range, and diameters up to 100 nm (Aqel et al., 2012). CNTs can be classified in terms of the number of walls as multi-walled carbon nanotubes (MWCNT) and single-walled carbon nanotubes (SWCNT). MWCNT is comprised of several layers of graphite surrounding a central tubule, whereas a SWCNT only has the central graphitic tubule. Fig. 1 - Molecular representation of MWCNT left and SWCNT right (Saifuddin et al., 2013) These two types of CNTs have been used to immobilize enzymes. SWCNTs are attractive for their higher surface area for enzyme interaction, but MWCNTs are desirable for their easier dispersibility and lower cost. Referring to their properties, carbon nanotubes generally have a large length-to-diameter aspect ratio of about 1000, so they can be considered as nearly one-dimensional structures (Dresselhaus et al., 2004). The strength of the sp2 carbon-carbon bonds gives carbon nanotubes amazing mechanical properties. Their densities can be as low as 1.3 g.cm-3 onesixth of that of stainless steel (Saifuddin et al., 2013). CNTs Young’s moduli are superior to all carbon fibers with values greater than 1 TPa which is approximately 5x higher than steel (Yu et al., 2000).The highest measured tensile strength was up to 63 GPa which is around 50 times higher than steel (Yu et al., 2000). Besides that, CNTs have good chemical and environmental stability and high thermal conductivity (3000 W.m-1.K-1). High-quality carbon nanotube materials are desired for both fundamental and technological applications. However there are four main challenges regarding synthesis of CNT: i) mass production, that is, the development of low-cost and large-scale processes; ii) selective production, that is, control over the structure and electronic properties; iii) organization, that is, control over location and orientation of the produced nanotubes on a flat substrate; Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 12 Amyloglucosidase Magnetic-SWCNTs Biofuel production (Goh et al., 2012) Horseradish peroxidase Maize-tassel-MWCNTs Metal ions biosensor (Moyo et al., 2014) Horseradish peroxidase MWCNTs H2O2 detector (Yamamoto et al., 2003) Horseradish peroxidase MWCNT/alumina-coated silica H2O2 detector (Huang and Tsai, 2009) Laccase Magnetic-MWCNTs Catechol biosensor (Pang et al., 2011) Laccase MWCNT-COOH Laccasebased biocatalysts (Silva et al., 2014) Thermomyces lanuginosus lipase Amino-MWCNTs Biocatalytic characterizat ion (Verma et al., 2013) 2.2 Enzyme Immobilization 2.2.1 General overview Enzymes can catalyze reactions in different states: as individual molecules in solution, in aggregates with other entities, and as attached to surfaces. The attached—or “immobilized”— state has been of particular interest for technical purposes. The term “immobilized enzymes” refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously” (Katchalski-Katzir, 1993). The introduction of immobilized catalysts has, in some cases, greatly improved both the technical performance of the industrial processes and their economy (Table 3). Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 13 Table 3 - Technological properties of immobilized enzyme systems Advantages Disadvantages Catalyst reuse Loss or reduction in activity Easier reactor operation Diffusional limitation Easier product separation Additional cost Wider choice of reactor Changes on enzyme properties Continuous process Interaction enzyme-substrate Enzyme stability No general method of immobilization During the past decades, immobilized enzyme technology has advanced into and everexpanding and multidisciplinary fields focused to analyze clinical, industrial and environmental samples. Enzyme immobilization has been used in different fields such as in medicine diagnosis and treatment of various diseases, antibiotic production, drug metabolism, food industry, biodiesel production, bioremediation, etc. (Khan and Alzohairy, 2010) The major components of an immobilized enzyme system are the enzyme, the matrix, and the mode of attachment of the enzyme to the matrix. The terms solid phase support, carrier, and matrix are used synonymously. The choice of the type of support influences the performance of the immobilized enzyme system. Ideal support properties include physical resistance to compression, hydrophilicity, inertness toward enzymes, ease of derivatization, biocompatibility, resistance to microbial attack, and availability at low cost (Brena and Batista-Viera, 2006). Table 4 presents the different types of supports, which can be classified as inorganic and organic according to their chemical composition. The organic supports can be further subdivided into natural and synthetic polymers (Cabral and Kennedy, 1991). Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 14 Table 4 - Classification of supports (Brena and Batista-Viera, 2006) Organic Natural materials  Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate  Proteins: collagen, albumin  Carbon Synthetic materials  Polystyrene  Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides and vinyl Inorganic Natural minerals: bentonite, silica Processed materials: glass nonporous and controlled pore, metals, controlled pore metal oxides The properties of immobilized enzyme preparations are governed by the properties of both the enzyme and the carrier material. The specific interaction between the latter provides an immobilized enzyme with distinct chemical, biochemical, mechanical and kinetic properties. Among the numerous parameters that have to be taken into account, the most important are outlined in Table 5. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 15 Table 5 - Selected characteristic parameters of immobilized enzymes (Tischer and Wedekind, 1999) Enzyme Biochemical properties molecular mass, prosthetic groups, functional groups on protein surface, purity inactivating/protective function of impurities Enzyme parameters specific activity, pH-, temperature profiles, kinetic parameters for activity and inhibition, enzyme stability against pH, temperature, solvents, contaminants, impurities Carrier Chemical characteristics chemical basis and composition, functional groups, swelling behavior, accessible volume of matrix and pore size, chemical stability of carrier Mechanical properties mean wet particle diameter, single particle compression behavior, flow resistance for fixed bed application, sedimentation velocity for fluidized bed, abrasion for stirred tanks Immobilized enzyme Immobilization method bound protein, yield of active enzyme, intrinsic kinetic parameters properties free of mass transfer effects Mass transfer effects consisting of partitioning different concentrations of solutes inside and outside the catalyst particles, external and internal porous diffusion; this gives the effectiveness in relation to free enzyme determined under appropriate reaction conditions Stability operational stability expressed as activity decay under working conditions, storage stability Performance productivity amount of formed product per unit or mass of enzyme enzyme consumption e.g. units kg–1 product, until half-life Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 16 2.2.2 Immobilization Methods Immobilization of macromolecules can be generally defined as a procedure leading to their restricted mobility. A classification of immobilization methods according to different chemical and physical principles is shown in Fig. 3. The most used enzyme immobilization strategies are also outlined in Fig. 4. Fig. 3 - Classification of immobilization methods (Tischer and Wedekind, 1999) Fig. 4 - Enzyme immobilization strategies: entrapment (a), encapsulation (b), support based (c) and self immobilization (d). Enzymes are represented by green circles (Brady and Jordaan, 2009) Enzyme entrapment (Fig. 4a) is typically achieved using a polymer network such as an organic polymer or sol–gel and is usually performed in situ. This technique has the advantage of protecting the enzyme thus preventing direct contact with the environment, minimizing the effects of gas bubbles, mechanical sheer and hydrophobic solvents. However, it has the disadvantage of mass transfer limitations and low enzyme loading (Lalonde and Margolin, 2008). A common method of entrapment is through use of silica sol–gel matrices formed by Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 17 hydrolytic polymerization. Bruns and Tiller, (2004) used this type of immobilization to immobilize horseradish peroxidase and chloroperoxidase in a polymer network. They have obtained an enzyme-complex network suitable for biocatalytic application in organic solvents. Encapsulation (Fig. 4b) is similar to entrapment. This technique protects the enzyme from the external environment but requires the use of large amounts of substrate, due to mass transfer limitations (Brady and Jordaan, 2009). Support based immobilization (Fig. 4c) consists on the immobilization of the enzymes on the surface of the different supports. According to the binding mode of the enzyme, the carrierbinding method can be further sub-classified into: physical adsorption; ionic binding and covalent binding (Goel, 1994). In other way, carrier-free enzyme immobilization (Fig. 4d) is possible using bifunctional cross-linkers, such as glutaraldehyde, to bind enzymes to each other without resorting to a support. Briefly, the physical adsorption mode is based on the adsorption of enzyme on the surface of water-insoluble carriers, mainly through Van der Walls interactions and hydrogen bonding. Hence, the method causes little or no conformational change of the enzyme or destruction of its active center. If a suitable carrier is found, this method can be both simple and cheap. However, it has the disadvantage that the adsorbed enzyme may leak from the carrier during use due to a weak binding force between the enzyme and the carrier (Goel, 1994). Another way to achieve immobilization at the carrier surface is by ionic binding. This method relies on the ionic binding of the enzyme protein to water-insoluble carriers containing ionexchange residues. Polysaccharides and synthetic polymers having ion-exchange centers are usually used as carriers. The binding of an enzyme to the carrier is easily carried out, and the conditions are much milder than those needed for the covalent binding method. Hence, the ionic binding method causes little changes in the conformation and the active site of the enzyme. Therefore, this method yields immobilized enzymes with high activity in most cases (Goel, 1994). The main difference between ionic binding and physical adsorption is that the enzyme-to-carrier linkages are much stronger for ionic binding although weaker than in covalent binding Finally, the covalent binding mode is based on the formation of covalent bonds between the enzyme and the support matrix. The functional groups that may take part in this binding are, for example, amino, carboxylic, phenolic, hydroxyl, etc. The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding. Therefore, covalent binding may alter the conformational structure and active center of the enzyme, resulting in major loss of activity and/or changes of the enzyme. However, the binding force between enzyme and carrier is so strong that no Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 18 leakage of the enzymes occurs, even in the presence of substrate or solution of high ionic strength (Goel, 1994). As shown before in Table 5 the choice of the most suitable carrier to perform immobilization is influenced by its chemical properties, mechanical stability and geometric properties. Hereupon, a wide range of synthetic carriers, organic or inorganic, can be created to encounter the optimal characteristics to perform immobilization of the desired enzyme. Thus, currently, there is an increasingly interest on using nano-dimensional materials as a support for the enzymatic immobilization, e.g. nanoparticles, nanofibres and, as used in this study, carbon nanotubes. 2.3 Peroxidase: origin, structure, function and applications Peroxidase (EC 1.11.1.7) is one of a number of enzymes that act as catalyst to allow a variety of biological processes. Peroxidases are a group of oxidoreductases that catalyze the reduction of peroxides, such as hydrogen peroxide, and the oxidation of a variety of organic and inorganic compounds (Johannes Everse et al., 1990). The term peroxidase represents a group of specific enzymes, such as NADH peroxidase (EC 1.11.1.1), glutathione peroxidase (EC 1.11.1.9), iodide peroxidase (EC 1.11.1.8), among others as well as a group of nonspecific enzymes that are simply known as peroxidases. They had attracted industrial attention because of its usefulness as a catalyst in clinical examinations and other applications. Peroxides are produced as byproducts of various biochemical reactions within organisms, but can cause damage as they are oxidizing agents. These enzymes break these compounds down in to harmless substances by adding hydrogen, obtained from another molecule — known as a donor molecule — in a reduction-oxidation (redox) reaction in which the peroxide is reduced to form water, and the other molecule is oxidized. There are a large number of peroxidases, and they are found in plants and animals, including humans. Perhaps, the best-known peroxidase is that from horseradish root (HRP) which, due to its broad specificity for hydrogen donors and its high catalytic efficiency, has been used widely in spectrophotometric determinations of biological materials. But currently, fungi (A. ramosus, C. cinereus) are known as new practical sources of peroxidase for industrial purposes (Shinmen et al., 1986; Morita et al., 1988). The peroxidase catalytic cycle involves distinct intermediate enzyme forms (Chung et al., 1997; Mantha et al., 2002). In the initial step, the native ferric enzyme is oxidized by hydrogen peroxide to form an unstable intermediate called compound I, which has a hem structure of Fe IV = O-porphyrin π-cation radical, with consequent reduction of peroxide to water (Eq. (2.1). Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 19 [ ( )]( ) → [ ( ) ]( ) (2.1) Then, Comp I oxidizes electron donor substrate (S) to give compound II, releasing a free radical ( ) (Eq. (2.2). Compounds I and II differ by only one electron on the porphyrin ring. [ ( ) ]( ) → [ ( )]( ) (2.2) Comp II is further reduced by a second substrate molecule, regenerating the iron III state and producing another free radical, according to the following equation: [ ( )]( ) → [ ( )]( ) (2.3) Several peroxidases have been isolated, sequenced and characterized. They have been classified essentially in three classes, depending on the organism: Class I, intracellular prokaryotic peroxidases, Class II extracellular fungal peroxidases and Class III secretory plant peroxidases. It has been proposed, from an extensive comparison among the amino acid sequences, that heme peroxidases from plants, fungi and bacteria are evolutionary related (Welinder, 1992). Production of peroxidase has also been achieved by genetic engineering approaches in Saccharomyces cerevisiae (Sawai-Hatanaka et al., 1995) and Asperguillus oryzae (Dalboge H. et al., 1992). It can be observed in Fig. 5 the main peroxidases superfamilies. Fig. 5 - Peroxidase classes (Fawal et al., 2013) In humans, and other mammals, a group of these enzymes called glutathiones, which contain the element selenium, are found both within and outside cells (Maier, 2014). Some of these catalyze reactions involving H2O2, while others use peroxide compounds of lipids fats and oils. Their main role seems to be to remove these potentially harmful oxidizing agents. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 20 Peroxidases in the saliva also enable redox reactions between H2O2 and chemicals called thiocyanates, producing compounds that can kill potentially harmful microorganisms (Maier, 2014). Thyroid peroxidase releases iodine from nutrients to form essential thyroid hormones. Like all enzymes, peroxidases are very large complex molecules with complicated shapes involving multiple folds. They come in a variety of types, some of which can use a wide variety of donor molecules and reduce a wide range of peroxides, and some of which are much more specific. Enzymes have an “active site,” which is the part of the molecule where the reaction takes place. This may be in an easily accessible part of the molecule, or it may be tucked away in a fold, where it can only be reached by a molecule of exactly the right shape. Fungal peroxidase is an example of an enzyme that can use a wide variety of donor molecules and peroxides. Molecular properties of the purified Arthromyces ramosus peroxidase (ARP) are summarized in Table 6. Table 6 – Molecular properties of a fungal peroxidase ARP (Nakayama and Amachi, 1999) Molecular weight (Da) 41,000 (Shinmen et al., 1986); 38,000 SDSPAGE (Welinder and Gajhede, 1993) Number of subunits 1 (Shinmen et al., 1986) Amino acids/subunit 344a,b (Kunishima et al., 1994) Disulfide bonds 4b pI 3,4-3,5 Optimum pH 6,0-7,0c, 5,0-8,0, 8,8-9,0d Km (μM) 52,6; 71,4 (Heinzkill et al., 1998) Optimum temperature 40 ºCc pH stability pH 5,0-9,0 at 30 ºC for 16 h Thermal stability Up to 50 ºC at pH 7.0 for 30 min Cofactor One protoheme IX/enzyme Absorption maxima 280, 415, 540, 640 nm oxidized form; 280, 438, 557, 585 nm reduced form Rz valuee 2,7 Sugar content and glycosylation sites 5%, Asn143 and Ser339b Metal ion requirement Two endogenous Ca2+ ionsb Cellular localization extracellular Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Literature Review 21 a From primary structure analysis. b From X-ray crystallography. c Oxidative coupling of phenols with 4-aminoantipyrine. d Chemiluminescent reaction with luminol. eRZ Reinheitszahl. value is the absorbance ratio with absorption at λmax of Soret band/absorption at 280 nm. In the past few years, several X-ray structures of peroxidases from different sources were reported: cytochrome-c peroxidase (CcP), reported more than 30 years ago (Finzel et al., 1984); lignin peroxidase (Piontek et al., 1993; Poulos et al., 1993; Neves, 2007); Arthromyces ramosus peroxidase (Kunishima et al., 1994); Coprinus cinereus peroxidase (CiP); (Petersen et al., 1994); manganese peroxidase (MnP); (Poulos et al., 1995) and pea ascorbate peroxidase (APX); (Patterson and Poulos, 1995). From a comparison among them, it was noted that, despite the low level of sequence homology (often <20%), the overall folding and the organization of the secondary structure is conserved (Poulos et al., 1995); In Fig. 6 it can be observed conservation of amino acids in peroxidase from different species. Fig. 6 - Conserved amino acid residues. The amino acid sequences near the invariant His and Arg residues of several peroxidases are aligned with each other. The proximal and distal His and essential Arg residues of ARP are indicated by ∆, о and □, respectively, above the sequences. The enzymes are as follows: ARP, A. ramosus peroxidase (Sawai-Hatanaka et al., 1995); BAP, lignin peroxidase of Bjekandera adusta (Kimura et al., 1991); PCP, lignin peroxidase of P. chrysosporium (Tien and Tu, 1987); MnP, manganese peroxidase of P. chrysosporium (Tien and Tu, 1987);; Turnip, turnip peroxidase (Welinder and Mazza, 1977); CCP, S. cerevisiae cytochrome c peroxidase (Kaput et al., 1982); and HRP, horseradish peroxidase (Welinder, 1976). The enzyme is divided in two different structural domains, enveloping the heme moiety. It has been proposed that the two domains originate from an early gene duplication event (Welinder and Gajhede, 1993). The structures of peroxidases are constituted by 10–11 αhelixes, linked by loops and turns, while β-structures are essentially absent or are a minor Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 28 120 kV, equipped with a 4 M pixel 2828 mm CCD camera from TRS at Centro de Química – Universidade de Trás-os-Montes e Alto Douro (UTAD). The morphology of the materials was observed using a high resolution (Schottky) environmental scanning electron microscope (SEM) with X-ray microanalysis and electron backscattered diffraction analysis (Quanta 400 FEG ESEM/EDAX Genesis X4M; secondary electron detector, 20 000, 15,00 kV) at the Materials Centre of the University of Porto (CEMUP). X-ray diffraction (XRD) analysis of selected samples was carried out at UTAD in a PANalytical X’Pert MPD equipped with a X’Celerator detector and secondary monochromator (Cu Ka  = 0,154 nm, 50 kV, 40 mA; data recorded at a 0,0178 step size, 100 s/step). Rietveld refinement with PowderCell software was used to identify the crystallographic phases present and to calculate the crystallite size from the XRD diffraction patterns. 3.5 Immobilization Technique In all experiments 4 mg of MWCNT were added to 1,2 mL of peroxidase solution (8 μLperoxidase/mLbuffer solution) under orbital stirring for 1h. After immobilization, MWCNT were washed several times with appropriated buffer. The immobilization yield (%) and the activity recovered (%) after immobilization are defined as: ( ) (3.1) ( ) ( ) (3.2) Where (U.L-1) is the enzymatic activity registered with a sample of solution of peroxidase prepared to perform immobilization, (U.L-1) the enzymatic activity registered with a sample of the supernatant recovered after performing immobilization, (U.g-1) the enzymatic activity registered with the MWCNTs onto which peroxidase was immobilized, (g) the mass of MWCNTs with enzyme immobilized and (L) the volume of reaction. In this work, the immobilization yield represents the yield of the immobilization process in terms of the quantity of active peroxidase adsorbed onto the carrier, while the activity recovered represents the yield of the immobilization process in terms of the capacity of the enzymes immobilized onto the carrier to perform catalysis. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 29 3.6 Enzymatic activity measurement The free and immobilized peroxidase activity were assayed spectrophotometrically (JASCO V- 560 UV-Vis spectrophotometer) with ABTS as substrate (0,4 mM) and H2O2 (0,33 mM) in 50 mM citrate/100 mM phosphate buffer at pH 4,5. To measure the free peroxidase activity, 100 μL of enzyme solution were mixed with 0,4 mL of ABTS and 1,4 mL of citrate/phosphate buffer (50/100 mM, pH 4,5, 40 ºC). The reaction was started by adding 100 μL of H2O2. The change in absorbance at 420 nm (ε = 36000 M-1 cm-1) was recorded automatically by the spectrophotometer and the catalytic activity was determined by measuring the slope of the initial linear portion of the kinetic curve. The free peroxidase activity is defined as: ⁄ (3.3) Where ⁄ is the quantity of enzyme capable of breaking down 1 µmol of ABTS per minute and per volume unit of enzymatic solution, ⁄ the absorvance per minute determined by linear regression, the dilution factor of the sample, the conversion factor from M to μM; and the molar extinction coefficient (36000 M-1 cm-1 at 420 nm). To measure peroxidase activity when using immobilized enzyme, MWCNT were mixed with 21 mL of citrate/phosphate buffer (50/100 mM, pH 4,5) at 40 ºC and 6 mL of ABTS, under magnetic stirring for 2 min. The reaction was started after adding 1,5 mL of H2O2 solution. Samples were taken every 20 seconds and absorbance was measured spectrophotometrically at 420 nm (Silva et al., 2014). After linear regression of the data obtained, enzyme activity was determined using Eq.(3.4: ⁄ (3.4) Where ⁄ is the quantity of enzyme capable of breaking down 1μmol of ABTS per minute and per mass unit of carrier, ⁄ the absorbance per minute determined by linear regression, the dilution factor of the sample, (L) the volume of reaction, the conversion factor from M to μM, the molar extinction coefficient (36000 M-1 cm-1 at 420 nm) and g the mass of MWCNTs with enzyme immobilized. 3.7 Optimization of the conditions for peroxidase immobilization on MWCNT Assays to determine the optimal peroxidase concentration were performed immobilizing the peroxidase solution in citrate/phosphate buffer (50/100 mM, pH 4,5) on 4 mg of MWCNTox- 900 at different concentrations of peroxidase: 3-11 μLperoxidase/mLbuffer for 1 h. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 30 To evaluate the influence of pH on peroxidase immobilization in 4 different carbon nanotubes, different buffers were used: citrate/phosphate 50 mM for pH 4,5; phosphate buffer 50 mM for pH 6,0, 7,0 and 8,0 and carbonate buffer 50 mM for pH 9,0. Measurements of enzymatic activity, according to the method described above, were performed for: i) initial peroxidase solution, ii) supernatants recovered after immobilization and iii) immobilized enzyme. To determine the optimum contact time, peroxidase immobilization was carried out several time laps ranging from 15 min to 4 h at the optimum conditions for this carrier (pH 4,5; 8 μLperoxidase/mLbuffer). 3.8 Thermal stability of free and immobilized peroxidase The thermal stabilities of the free and immobilized peroxidase were investigated by incubating the free and immobilized enzymes in citrate/phosphate buffer (50/100 mM, pH 4,5) and phosphate buffer (50 mM, pH 6,0), respectively, at different temperatures (30-50 ºC). Immobilized peroxidase was suspended in 500 μL of the immobilization buffer. In both cases samples were removed regularly from the water bath and enzymatic activity was quickly determined according to the methods described above. The thermal parameters were calculated according to a simplified deactivation model described in the literature (Henley and Sadana, 1985): → → (3.5) [ ] [ ] (3.6) Where (%) is the residual enzyme activity, and ratios of specific activities (remaining activities), respectively to the different states E1/E and E2/E (see Eq. 3.5), (h-1) and (h-1) the thermal inactivation rate constants and (h) the time. Analyzing the data obtained, it was considered that peroxidase undergoes a conformational transition due to the increase in temperature according to the model of Eq. (3.5. This implies that inactivation follows a single exponential decay, in which and , leading to: [ ] (3.7) The thermal parameters and of the model described in Eq. (3.7 were estimated by a nonlinear fitting of the experimental data, using CurveExpert v 2.0.4 © 2011-2014. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 31 Biocatalyst half-life ( ⁄) was calculated from Eq. 3.7, using the estimated parameters and and making equal to 50. The energetic barrier (activation energy, ) for the thermal deactivation process was calculated by nonlinear regression considering the Arrhenius equation, defined as: (3.8) Where (h-1) is the pre-exponential factor, (J mol-1) the energy of activation, (K) the temperature and the universal gas constant (8,314 J mol-1 K-1). The standard free energy for the thermal inactivation ( ) was calculated from the firstorder rate constant of inactivation process, at different temperatures, using the following equation (Longo and Combes, 1999): ( ) (3.9) Where (J h) is the Planck constant (1,84E-37) and (J.K-1) the Boltzmann constant (1,3807E-23) The activation enthalpy ( ) was calculated from the activation energy: (3.10) And the activation entropy ( ) was calculated from the enthalpy and standard free energy as follow: (3.11) 3.9 Determination of kinetic parameters of free and immobilized peroxidase To determine the kinetic parameters of free peroxidase, enzymatic activity was measured for concentrations of ABTS ranging from 3 to 120 μM, according to the method described previously. Similarly, the kinetic parameters of immobilized peroxidase were determined measuring the enzymatic activity, for the catalysis of ABTS at concentrations ranging from 0,01 to 0,9 mM. The hydrogen peroxide concentration was kept at 0,33 mM. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 32 Since the data obtained followed the pattern predicted by the Michaelis-Menten model (Eq. 3.12), CurveExpert (v2.0.4 © 2011-2014) was used to perform a non-linear fit of the experimental values in order to determine and . [ ] [ ] (3.12) Where (mM.min-1) is the catalytic reaction rate, (mM min-1) the maximum rate of the reaction, [ ] the concentration of substrate (mM) and the Michaelis-Menten constant. (Steevensz et al., 2009). 3.10 Storage and operational stability of immobilized peroxidase The stability of free and immobilized peroxidase was evaluated at 4 ºC, for one month. Peroxidase was immobilized on MWCNT in phosphate buffer (50 mM, pH 6,0), according to the method described above. The operational stability was determined by reacting peroxidase immobilized on mMWCNT with 6 mL of ABTS (0,4 mM) in 21 mL of citrate-phosphate buffer pH 4,5 at 40 ºC. The reaction was started by adding 1,5 mL of H2O2 (0,35 mM). At each cycle, a sample was withdrawn in 30 sec intervals over 4 minute period and the absorbance was measured at 420 nm. The mMWCNT-Peroxidase conjugates were then removed by a simple magnet, washed once with phosphate buffer (50 mM, pH 6,0), and re-immersed in a fresh ABTS solution to begin the next cycle. A total of 9 cycles were performed. The operation stability of the immobilized peroxidase on a MWCNT membrane and on MWCNTox-400 was also analyzed, as described as Appendix information. 3.11 Protein determination The supernatants from the immobilization method were collected and used in the Bradford protein assay (Appendix 1) to determine the mass of peroxidase on 4 mg of MWCNTs. This was done by measuring the difference in the amount of enzyme in the supernatant and the total amount of enzyme added to the carbon nanotubes. This mass balance was also estimated for the second wash and thermal incubation (50 ºC) supernatants. 3.12 Gel electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in order to evaluate the purity of commercial peroxidase solution. The gel was then stained with Coomassie Blue stain for 24 h and destained using acetic acid to visualize the separated enzyme (Appendix 1) Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 33 3.13 Discoloration of textile dyes Degradation of 3 dyes (Reactive Black 5, Reactive Blue and Bromophenol Blue) was evaluated incubating free or immobilized peroxidase on mMWCNT in solutions of each one of the dyes in test, for 24 h. More details about these dyes can be consulted in Appendix 1. The reaction solutions (10 mL) were prepared dissolving a stock solution of the dye in citrate/phosphate buffer (50/100 mM, pH 4,5). Absorbance of all solutions was measured spectrophotometrically, before ( ) and after ( ) the incubation period, at the maximum absorption wavelength of each dye: RB5 (590 nm), BPB (594 nm) and RB (590 nm). Controls with mMWCNT without peroxidase and with inactivated peroxidase (5 min, 100 ºC) were made to evaluate the dye elimination by adsorption. For each dye solution, the discoloration efficiency is defined as: (3.13) Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Experimental Section 34 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 35 4 Results and Discussion 4.1 Characterization of MWCNT The development of an efficient functionalized nanotubes support for peroxidase immobilization, using a non-covalent binding technique for investigating its influence on the biocatalyst structure function relationships, was one of the objectives of this work. In this way, multi-walled carbon nanotubes (MWCNTs) represent ideal nanoscale supports due to their easy acid oxidation to produce hydrophilic carboxylic acid and hydroxyl groups along their sidewalls. Moreover, their high surface areas may lead to high functional enzyme loadings, while their high thermal and mechanical stability make them amenable for use as robust supports for protein attachment (Asuri et al., 2006). Thus, in order to study the interaction of peroxidase with the surface of MWCNT and assess its importance on peroxidase immobilization yield and respective activity, oxygen containing groups were selectively introduced or removed on MWCNTs side walls by liquid-phase oxidation and thermal treatments, as described in the experimental section. 4.1.1 N2 adsorption-desorption isotherms Nitrogen adsorption isotherms were carried out for the obtained samples and the results are presented in Fig. A1. The characterization of these materials is summarized on Table 7. Table 7 - Specific surface area , pore volume and pore diameter for the different MWCNT samples. Sample (m2.g-1) (cm3.g-1) (nm) CO (µmol.g-1) CO2 (µmol.g-1) CO+ CO2 (µmol.g-1) CO/ CO2 MWCNT 255 0,944 3,2 7,2 348 70 418 5,0 MWCNTox 276 1,073 15 3,0 800 412 1212 1,9 MWCNTox -400 291 1,154 20 4,0 623 223 846 2,8 MWCNTox -900 301 1,247 25 6,9 176 37 213 4,8 Two ranges of different behavior can be observed in Fig. A1 as relative pressure (p/p0) increases. For low pressures (0<p/p0<0,4) adsorbed volume increases slowly, indicating monolayer completion. The absence of a steep increase at very low p/p0 reflects the absence Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 36 of micropores with sizes in the order of magnitude of the nitrogen molecule (Vicente et al., 2011). For pressures corresponding to (0,4<p/p0<1,0), hysteresis loop can be observed, which can be related to capillary condensation, which is known to occur in mesopores (2-50 nm) (Gregg, 1982), precisely the order of magnitude of the inner cavities of these MWCNTs samples, as shown in Table 7. The N2 adsorption uptake near p/p0=1,0 is explained by the presence of larger pores on the surface of MWCNT as a result of acid oxidation and thermal treatments. Aggregates of CNT were found to cause such effect in previous studies (Yang et al., 2001). The surface areas of the samples, calculated by the BET method ( ) revealed that the oxidation treatment lead to an increase of the surface area of the resulting material. After the reflux of pristine MWCNT in HNO3, the surface area increased approximately 8% (MWCNTox, Table 7). The total pore volume ( ) and respective diameter ( ) were also increased, wherein the pore diameter of MWCNTox was around 4.7 times higher than that of pristine MWCNT. This happens because this oxidative process occurs under strong acidic conditions, creating defect sites ends and/or holes on CNTs sidewall. These findings are in line with published works for nitric acid oxidation (Silva et al., 2014) and for ozone oxidation pre-treatment (Liu et al., 2009). 4.1.2 Temperature programmed desorption (TPD) TPD was used to analyze the oxygen functional groups of carbon nanotubes material. This investigation could offer valuable detailed information about the chemical nature of catalyst surface, since the different surface oxygenated groups could be thermally decomposed by at specific temperatures (desorption temperature) turning possible its identification from the TPD spectra. The main possible functionalities in an individual graphene sheet are schematically summarized in Fig. A2. It should be noticed that the difference of desorption temperatures of some functional groups in literature could be high, as shown in Table A1. Carboxylic groups are decomposing at low temperatures, anhydrides at intermediate temperatures and lactones at high temperatures. As mentioned before, oxidation is an easy method to improve the oxygen amount on the surface of CNTs. The TPD profiles of purified MWCNTs, oxidized MWCNTox and calcined MWCNTox-400 and MWCNTox-900 are displayed in Fig. 10. The total amounts of CO and CO2 evolved from the samples are also presented in Table 7. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 37 Fig. 10 – CO2 (a) and CO (b) TPD profiles of pristine and functionalized MWCNTs. As expected, the surface of MWCNTox was highly functionalized. In the TPD CO2 profile, a larger peak at 300 ºC and others at 500 ºC and 650 ºC were identified, assigned to the decomposition of carboxylic acid, anhydride and lactone, respectively (Liu, 2008). The peak observed at 750 ºC on CO profile of this sample corresponded to the decomposition of quinone groups. After calcination at 400 ºC, a CO2 desorption peak at about 650 ºC was observed which indicates that the thermal treatment at 400 ºC remove part of carboxylic acids, but some carbonyl/quinones remains on the surface of the MWCNTox-400. When further treatment at 900 ºC, few oxygenated surface groups were anchored in carbon nanotubes. It 0,00 0,02 0,04 0,06 0,08 0,10 0,12 100 300 500 700 900 CO2 (umol.g-1) T (ºC) (a) 0,00 0,05 0,10 0,15 0,20 100 300 500 700 900 CO (umol.g-1) T (ºC) MWCNT MWCNTox MWCNTox-400 MWCNTox-900 (b) Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 44 dependence of the activity of the immobilized peroxidase on solution pH is expected to be different from that of the free peroxidase. The immobilization yield (%) and the recovered activity (%) are shown in Table 8 and Table 9, respectively. These two parameters allow us to analyze the effectiveness of each type of support for the immobilization of peroxidase at different pH values. Table 8 – Peroxidase immobilization yield on the MWCNT materials for the different pH Immobilization Yield % pH MWCNT MWCNTox MWCNTox-400 MWCNTox-900 4,5 99,8 87,9 83,7 94,0 6 96,4 75,5 73,2 85,1 7 98,4 68,7 67,2 83,4 8 90,8 68,8 65,3 80,3 9 95,4 73,3 47,8 84,8 Table 9 - Recovered activity of the immobilization process for the different pH of the medium used to perform immobilization Activity Recovered % pH MWCNT MWCNTox MWCNTox-400 MWCNTox-900 4,5 2,0 2,3 3,0 2,3 6 2,3 1,8 2,9 2,3 7 1,2 0,9 1,6 2,4 8 1,8 0,7 1,0 2,2 9 1,4 0,4 0,8 1,6 It can be observed that even without any further treatment, purified MWCNT shows higher immobilization capacities (90-100%) over the range of pH tested. This value was also obtained for laccase immobilization (Silva et al., 2014). In this purification treatment, no defects were created at the surface of CNTs. So, peroxidase is immobilized on MWCNT by physical adsorption, where hydrophobic and π-π stacking interactions (with peroxidase hydrophobic regions and aromatic rings, respectively) are the most probable interactions. In other way, the peroxidase immobilization on functionalized MWCNT results from specific electrostatic interactions between carboxyl groups and proper polar or ionic groups of peroxidase. This should result in much higher immobilization yield, but this did not occur. It Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 45 can be related to the creation of covalent bonds between the COOH groups and the enzyme, which is not likely to occur in this case; being necessary a mediator such as carbodiimide for carboxylic acid activation (Asuri et al., 2006). Among the modified materials, MWCNTox-900, which has a normally neutral, presents the highest immobilization capacity. In this case, more intense electrostatic interactions are expected for acid-neutral pH values, conditions at which the carbon support is positively charged and the enzyme is negatively charged. On the other hand, MWCNTox-400 shows the lowest immobilization efficiency, which may be related to the proximity between the of the carbon materials (4,0) and the pI of peroxidase (3,5). The same happens with MWCNTox, which has a of 3,0. However, it shows higher immobilization yield than MWCNTox-400. These results can be explained by the fact of MWCNTox surface being negatively charged while peroxidase is positively charged, leading to the existence of stronger electrostatic interactions between the enzyme and this materials, rather than with MWCNTox-400. It must be noticed that even if the immobilization efficiency is high, that doesn’t mean that all enzymes immobilized are available to react, since they can attach to enzymes previously attached to the carrier or in an orientation that block the active site of the enzyme or too close to other enzyme molecules, generating allosteric hindrances that difficult/prevent the access to the substrates. Then, according to Table 9, considering the values between the optimal range of pH 4,5-7, the maximal recovery of activity is registered on carrier with a thermal treatment at 400 °C (3,02%), despite the immobilization efficiency has not been the best. This may be due to the arrangement of the enzymes on the nanotubes surface, where the density and the orientation of the enzymes optimize their activity. An example that can support this explanation is the case of purification with H2SO4. In this case, the immobilization yield is higher, but the activity recovered was one of the lowest. Possible allosteric changes in enzyme may have happened. It is worth noticing that the percentage of recovery of activity is also low. In consequence, the majority of the enzymes immobilized on the different carriers are not able to perform catalysis. Considering all the results, we can say that the best pH value to perform the enzymatic immobilization is 4.5. Regarding the choice of support, if we want a better enzyme activity we should choose the treatment at 900 °C. On the other hand, if we want perform immobilization without wasting much enzyme, we should choose the nanotubes purified with H2SO4. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 46 4.4 Thermal stability of free and immobilized peroxidase The temperature influences the speed of an enzymatic reaction such as with any chemical reaction. However, in the case of enzymes, the effect produced is more complex due to external factors such as the effect of temperature on enzyme structure and on complex enzyme-substrate, ionization of aminoacids, etc. The reaction rate typically reaches a maximum temperature, and for higher temperature values, there is a rapid loss of enzymatic activity resulting from protein denaturation. The enzyme deactivation during reaction and storage (section 4.6) is often a critical limitation to commercial use. Therefore, the thermal stability of free and immobilized peroxidase (MWCNTox-400) was evaluated. The samples were incubated in buffer solution at different temperatures from 30 to 50ºC. The thermal parameters for immobilized peroxidase on purified and functionalized MWCNTs were also investigated at 40 and 50ºC. It was considered that the pattern of thermal deactivation of this enzyme follows an exponential decay, so, the experimental points for both free and immobilized peroxidase could be adequately represented by Equation (3.7. In this model the enzyme was inactivated in only one phase (a one-step transition between the active and denatured state) considering the possibility of the existence of remaining activity, which is represented by the α parameter. As can be seen in Fig. 13, it is evident that the thermal stability of the peroxidase immobilized on CNTs was improved. The free peroxidase lost its activity after 40 min of incubation at 50ºC and it was nearly zero after 2 h at 35-40ºC, whereas the immobilized peroxidase retained almost 44% of its initial activity after 2 h of incubation at highest temperature. This result is in agreement with thermal stabilization shown in Table 6 for other fungal peroxidase. This inactivation is probably due to a higher vibration of the peroxidase structure that may break some chemical bonds inside the molecule and, as consequence, change its 3D structure (Silva et al., 2014). Thus, the enzyme acquires a conformation which doesn’t allow them to maintain their catalytic capacity. For higher temperatures, (Asuri et al., 2006) shown that the native SBP has a half-life of 9 min at 90 ºC and this is increased sevenfold for the MWCNT-SBP conjugates. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 47 Fig. 13 - Thermal inactivation of (a) free peroxidase and (b) immobilized on MWCNTox-400 The increase thermal stability observed for the immobilized enzyme observed may be attributed to a reduction in the protein structure mobility, due to anchorage to the support and subsequent translation of the rigidity at each anchorage point to the whole enzyme structure, thus protecting it from the denaturing effects of the environment (Verma et al., 2013). Specifically, the native enzyme has a half-life of 5.6 min at 50 ºC (Table 10). The stabilization of peroxidase upon conjugation onto the MWNCTs is not surprising given the well-known stabilization of enzymes on solid supports. The increased stabilization can be due to multi-point attachment of the enzyme to the support and/or because of decreased 0 20 40 60 80 100 020 40 60 80 100 120 Peroxidase activity (%) Time (min) 30 ºC 35 ºC 40 ºC 50 ºC 0 20 40 60 80 100 020 40 60 80 100 120 Peroxidase activity (%) Time (min) 35 ºC 40 ºC 45 ºC 50 ºC (b) (a) Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 48 protein–protein interactions on the highly curved surface of carbon nanotubes (Asuri et al., 2006). The thermal parameters , and ⁄ for free and immobilized peroxidase are presented in Table 10. Table 10 - Thermal parameters and thermal stabilities obtained for the thermal inactivation of free and immobilized peroxidase Free peroxidase T (ºC) k (h-1) α t1/2 (h) ΔG0 (kJ.mol-1) ΔH (kJ.mol-1) ΔS (J.mol-1) Ea (kJ.mol-1) 30 1,316±0,081 8,135±2,617 0,597 94,239 71,661 -74,477 74 35 1,479±0,117 4,162±4,039 0,442 95,536 71,620 -77,612 40 3,039±0,094 2,480±1,264 0,237 95,254 71,578 -75,605 50 7,323±0,228 0,867±1,099 0,093 96,017 71,495 -75,883 Immobilized peroxidase T (ºC) k (h-1) α t1/2 (h) ΔG0 (kJ.mol-1) ΔH (kJ.mol-1) ΔS (J.mol-1) Ea (kJ.mol-1) 35 2,115±1,204 76,824±4,598 - 94,620 119,129 79,535 122 40 2,124±0,580 44,290±6,238 1,072 96,187 119,087 73,131 45 6,733±1,061 44,569±1,921 0,345 94,712 119,046 76,485 50 16,848±4,217 41,366±2,070 0,114 93,778 119,004 78,063 The stabilization of peroxidase through this immobilization method can be also confirmed by these results. After the immobilization, higher enzyme half-life time ( ⁄) was obtained, which means that a longer period of time is necessary to denaturate/deactivate the Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 49 peroxidase. For example, comparing at 40 ºC, the ⁄ was 4.5 times higher for immobilized peroxidase than for the free form. Although in some cases initial inactivation is faster for immobilized peroxidase 50 ºC, which results in a higher k parameter, the remaining activity, parameter, increases after 2 hours. This fact is rapidly confirmed by the Fig. 13, where the remaining enzyme activity was considerably higher for immobilized peroxidase at all temperatures. As previously stated, the CNTs adsorb the enzyme and protected it from the denaturing effects of the environment. The Arrhenius activation energy (Ea) is best regarded as an experimentally determined parameter that indicates the sensitivity of the reaction rate to temperature. Thus, the activation energies, for the thermal enzymatic deactivation, were obtained by Equation (3.8 As a non-linear equation, the Ea parameter was estimated by an Arrhenius plots (ln k vs T-1). The results are shown in Fig. A8. The activation energies of free and immobilized peroxidase were 74 and 122 kJ.mol-1, respectively. These results confirm what was been said so far, as more energy is required to deactivate the immobilized peroxidase. The thermodynamic parameters presented in Table 10 can be helpful in predicting enzyme stability. Protein unfolding is followed by the rupture of several links, generating a disorganized system. This increase in entropy ( ) (Eq. (3.11) is compensated by a decrease in Gibbs free energy (Eq. (3.9), making it easier for denaturation to occur, meaning that, when the susceptibility to denaturation is higher, the enzyme is in a less energetic state and in greater disorder, since its original structure has been destroyed (Aguiar-Oliveira and Maugeri, 2011). In relation of free peroxidase, the values of enthalpy of denaturation ( ) and free energy of denaturation ( ) were 71,578 kJ.mol-1 and 95,254 kJ.mol-1 while that of entropy of deactivation ( ) was -75,605 J.mol-1.K-1 at 40 ºC. At this temperature, for immobilized enzyme, the value of was slightly higher 96,187 kJ.mol-1. This increase in revealed that the thermal stabilization of peroxidase was due to the higher free energy (functional energy) which enabled the enzyme to resist against unfolding of its transition state. It should be noted that all values was positive and in the same order of magnitude, which indicates that the thermal inactivation process is thermodynamically nonspontaneous and slightly decreases with the temperature. The large value of is associated with increased enzyme stability unless coupled with a large compensatory increase in the value of , which is destabilizing. Hence, stability of enzyme is enhanced with moderately high levels of and low levels of . When a protein molecule is deactivated the randomness of the system increases which is a direct measure of entropy. By second law of thermodynamics, the entropy of the system is positive but the negative values of entropy are often encountered in the case of enzymes. As shown in Table Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 50 10, a large values of were obtained for immobilized peroxidase, which indicates more stability. However, positive values were obtained for immobilized peroxidase. The positive values of indicates protein unfolding reaction as the rate-limiting step during thermal deactivation under the studied conditions (Owusu et al., 1992). Thus, the negative for free peroxidase suggests that the rate-limiting reaction probably involves the aggregation of partially unfolded enzyme molecules which are predominant during the exposure of protein to high temperatures. Finally, in order to evaluate the influence of carbon nanotubes functionalization in the thermal stability of peroxidase, the thermal deactivation parameters were estimated at 40 ºC and 50 ºC, represented in Table A4 and Table 11, respectively.. Table 11 – Thermal parameters (50 ºC) for free and immobilized peroxidase on purified and functionalized MWCNT Enzyme Thermal parameters (50ºC) t1/2 (h) α k (h-1) Free -0,867±1,099 7,323± 0,228 0,093 MWCNT 16,028±3,133 7,691±1,263 0,118 MWCNTox 29,876±3,027 13,777±3,691 0,091 MWCNTox-400 41,366±2,070 16,848±4,217 0,114 MWCNTox-900 28,686±2,850 10,097±2,146 0,120 The data show that the peroxidase is thermally more stable when it is immobilized on MWCNTox-400, at 50 ºC. In this case, the parameters α and k are higher compared to other samples, (41,366 and 16,848 h-1) which means that despite the faster initial inactivation, after 2 h of incubation period, peroxidase maintains higher residual activity (41,37%). This indicates that the adsorption of peroxidase is more stable in this support due to the availability of OH groups on the surface of the CNTs, which allows the creation of hydrogen bonds with the enzyme. Comparing the purified MWCNTs with the sample MWCNTox, it can be said that the oxidation treatment can promote the stabilization of immobilized enzyme. The α value was almost 2 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 51 times higher for MWCNTox. A sample with same treatment was used to immobilize the laccase enzyme, and it was obtained a α value 1.3 times higher than purified MWCNTs. This can be explained by the presence of carboxylic acid and phenol groups on the nanotube’s surface, which provides a more stable peroxidase immobilization. On the other hand, MWCNTs have negligible amounts of surface groups, so the interactions between this support and the peroxidase are weaker. The value of this parameter should also be lower for nanotubes treated at 900 ºC, almost equal to that of the purified nanotubes, but this was not observed. This is probably due to intense electrostatic interactions and to the higher surface area and higher porous diameter than pristine MWCNTs, which may protect the enzyme from denaturing effects of the environment. 4.5 Kinetic parameters of free and immobilized peroxidase A more accurate assessment of the changes in the peroxidase enzyme after immobilization would be to examine the difference in enzyme kinetics, i.e. the affinity of ABTS oxidation. To obtain the kinetic parameters and , the initial reaction rates of the ABTS oxidation by both free and immobilized peroxidase were measured at a predetermined range of substrate concentrations. The parameters for free and immobilized peroxidase were calculated using the Michaelis-Menten equation as shown in Eq. (3.12 and are summarized in Table 12. 0,0 0,5 1,0 1,5 2,0 2,5 020 40 60 80 100 120 140 V0(μM.min-1) [ABTS] μM Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 52 Fig. 14 – Michaelis-Menten plot for peroxidase catalysis. Effect of substrate concentration on the initial rate of oxidation of ABTS catalyzed by (a) free peroxidase and (b) MWCNT- peroxidase. The behavior of free and immobilized peroxidase was different. Free peroxidase followed conventional Michaelis-Menten kinetics for ABTS as substrate (Fig. 14a) with a of 0,033±0,004 mM and a of 2,979±0,130 mM.min-1. This value of obtained is in accordance with data on Table 6 for Coprinus cinereus peroxidase. On the other hand, for MWCNT-peroxidase conjugates (Fig. 14b), the kinetic parameters and were 0,064±0,018 mM and 2,303±0,137 mM.min-1, respectively. The constant measures the affinity of enzyme-substrate complex and it is specific for each enzyme. Thus, the higher , the more amount of substrate is necessary, for the reaction, to reach half of , that is, the affinity of the enzyme for the substrate is smaller. Then, it can be concluded that the immobilization diminishes the affinity of peroxidase to ABTS, however diffusional limitation should also be considered. As seen in Table 12, the catalytic efficiency, as given by the ⁄ ratio, has decreased after immobilization onto purified MWCNTs. This is expected, as immobilization of the peroxidase enzyme would induce changes in the enzyme conformation that, directly or indirectly, affect the active site, thus reducing the enzyme activity. In the same way, the immobilization process induces some limitations of mass transfer of the substrate to the surface of MWCNTs. In other works, the catalytic efficiency of SBP covalently immobilized on MWCNTs was nearly 40% of that for native SBP in aqueous solution (Asuri et al., 2007) and up to 57% for amyloglucosidase (AMG) immobilized noncovalently on SWCNTs (Goh et al., 2012). The enzyme activity after covalent immobilization is much lower than physical adsorption. (Goh et al., 2012) deduce that that the increased 0,0 0,5 1,0 1,5 2,0 2,5 0,0 0,2 0,4 0,6 0,8 1,0 V0 (mM.min-1) [ABTS] mM Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Results and Discussion 53 enzyme loading in covalent immobilization is offset by a greater change in the enzyme conformation, therefore resulting in reduced catalytic activity. Table 12 – Kinetic parameters of ABTS oxidation for free and immobilized peroxidase on purified MWCNTs Peroxidase (mM.min-1) (mM) ⁄ (s-1) Free 2,979±0,130 0,033±0,004 1,505 Immobilized 2,303±0,137 0,064±0,018 0,600 Several methods have been used to determine the changes on secondary structure of enzymes after immobilization on carbon nanotubes, e.g. circular dichroism spectroscopy CD 1 and FT-IR spectra (amide I region 1600-1700 cm-1). This region, which consists mainly of the C=O stretching vibration of the backbone peptide bonds in proteins, has been used to estimate quantitatively the secondary structural features of proteins (Dong et al., 1990; Vedantham et al., 2000). This spectrum can be used to obtain the α-helix and β-sheet contents of peroxidase. Karajanagi et al., (2004) reported that the differences in secondary structure between the soluble and adsorbed SBP, as represented by the simple sum of magnitudes of changes in α-helix and β-sheet contents, were 13% and, relatively to the 3D shape, the SBP retains its native three-dimensional shape on the nanotube surface. Other case, when HRP is attached covalently to SWCNTs retains 68% of its native α-helix (Asuri et al., 2007); this value is similar to that for the previous case, where SBP retains 77% of its native α-helix content when noncovalently adsorbed onto SWCNTs (Karajanagi et al., 2004). Goh et al., (2012) studied the role of the surface chemistry of carbon nanotubes on the enzyme conformation change. They concluded that the structural changes by physical adsorption and covalent approach are similar probably because both surfaces involve carboxylic groups in the enzyme immobilization, thus resulting in similar structural changes (Goh et al., 2012). 4.6 Storage and operational stability of immobilized peroxidase The stability of a product is an important consideration in the estimation of its shelf life and can affect its feasibility for apply in the industry or other commercial use. The activity of the immobilized peroxidase should be stable for a reasonable period of time, before any application. Hence, the storage stability of free and MWCNT-peroxidase conjugates was 1 CD analysis is considered more accurate, as the protein remains in its aqueous environment during the analysis, and thus can be used to correlate to its activity Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Conclusion 60 - The maximum peroxidase activity was obtained when immobilization was performed at pH 6. It can be assumed that the immobilized peroxidase is stable at relatively acidic conditions; - Different MWCNT materials presented different behaviors along the immobilization pH range tested. The highest enzymatic activity value obtained was 327.4 U.g-1, at pH 6, for the CNTs heat treated at 900 °C; - Purified MWCNT allows a better enzymatic activity than those oxidized with nitric acid (MWCNTox); - Purified MWCNT shows higher immobilization capacities (90-100%) over the range of pH tested; - Concerning the choice of the support, the carrier with better immobilized enzyme activity is the one which was treated at 900 °C and the carrier with better enzyme immobilization yield is the one which was purified with H2SO4; - The optimum conditions for peroxidase immobilization on MWCNTs are acid pH (4.5), peroxidase concentration of 8 µL/mLbuffer and contact time of 30 min; - On the topic of the thermal treatment, the free peroxidase lost its activity after 40 min of incubation at 50ºC and it was nearly zero after 2 h at 35-40ºC, whereas the immobilized peroxidase retained almost 44% of its initial activity after 2 h of incubation at the highest temperature. For example, comparing at 40 ºC, the ⁄ was 4.5 times higher for immobilized peroxidase than for the free form; - Peroxidase is thermally more stable when it is immobilized on MWCNTox-400 at 50 ºC, due to the availability of OH groups on the surface of the CNTs, which allows the creation of hydrogen bonds with the enzyme.; - The activation energies of free and immobilized peroxidase were 74 and 122 kj-mol-1, respectively. These results confirm that further energy is compulsory to deactivate the immobilized peroxidase; - The oxidation treatment promotes the stabilization of immobilized enzyme and the α parameter was almost 2 times higher for MWCNTox samples; - The kinetic behavior of free and immobilized peroxidase was different. Free peroxidase followed conventional Michaelis-Menten kinetics for ABTS as substrate with a of 0,033±0,004 mM and a of 2,979±0,130 mM.min-1 and for the immobilized, the values were 0,064±0,018 mM and 2,303±0,137 mM.min-1, respectively; Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Conclusion 61 - The immobilization decreases the affinity of peroxidase with the substrate, however diffusional limitation should also be considered; - The activity of the peroxidase was more stable when stored in the immobilized form than at free form; After 40 days, the MWCNT-peroxidase conjugates retained ca. 34% of its initial activity, whereas native peroxidase retained only 5% of its activity; - Despite the mMWCNT-peroxidase conjugates can be effortlessly recovered with a simple magnet, the peroxidase loses almost all of its activity after the first cycle. The same behavior was observed with peroxidase immobilized on MWCNTox-400; Regarding the biocatalyst application: - Peroxidase can be used for dye discoloration, obtaining a dye discoloration of 23% for RB5, 91% for RB and 98% for BR; - Immobilization process reduces the dye discoloration percentage because of diffusional limitations. However, the magnetic support may be beneficial due to its inexpensive and easy reuse; Overall, the advantages of the using MWCNT supported were demonstrated, as well as the importance of support functionalization in the protection of peroxidase structure perspective. Therefore, the usage of this enzyme by the industries is thought to be a massive advantage. 5.2 Perspectives for further research During this work, it was noticed that more assessments and more information would be interesting about the theme addressed in this dissertation. Therefore, there are several lines of research which should be pursued. First of all, although studies on the influence of the functionalization of MWCNTs in the peroxidase immobilization have been performed and the optimal immobilization conditions being established, it was noticed that it was difficult to reuse the MWCNTs-peroxidase complexes. Thus, the covalent immobilization of peroxidase on MWCNTs could be examined in future works. This analysis will allow the comparison of these two types of immobilization and it is expected that the peroxidase is linked more strongly to MWCNTs would facilitate its reusability. Another aspect, which may also be related to the previous problem is to study the change of the three dimensional structure of peroxidase, after immobilization on these samples, by circular dischroism spectroscopy. This method allows determining the change in α and β conformation of the enzyme after utilization in the immobilized form. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Conclusion 62 Regarding the magnetization process, it can be done in future works another type of insertion of MNPs on MWCNTs. In this thesis, it was used the covalent linkage between MNPs and the carboxylic groups on the surface of MWCNTs, through the use of binders such as APTS. However, it will be possible to make the insertion of MNPs inside the MWCNTs leaving the entire surface for peroxidase immobilization. This will enable any functionalization on its surface, adding the same magnetic characteristic of the carrier. As a final point, concerning the application of this biocatalyst and due to time constrains, the conditions used for dye degradation should be optimized. 5.3 Final appreciation In brief, this project has been considered as a very successful experience, in which all the expectations were achieved. 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Hoffman (1996). "Surface-oxidized carbon fibers: I. Surface structure and chemistry." Carbon 34(8): 983-998. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 77 Appendix 1 1.1 Characterization of MWCNT 1.1.1 N2 adsorption-desorption isotherms 0 200 400 600 800 1000 00,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 Vads (cm3 g-1, STP) p/p0 MWCNT 0 200 400 600 800 1000 1200 0 0,2 0,4 0,6 0,8 1 Vads (cm3 g-1, STP) p/p0 MWCNTox Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 78 Fig. A1 - Nitrogen adsorption-desorption isotherm of obtained samples. Temperature was fixed at -196 ºC. 0 200 400 600 800 1000 1200 0 0,2 0,4 0,6 0,8 1 Vads (cm3 g-1, STP) p/p0 MWCNTox-400 0 200 400 600 800 1000 1200 1400 0 0,2 0,4 0,6 0,8 1 Vads (cm3 g-1, STP) p/p0 MWCNTox-900 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 79 1.1.2 Temperature programmed desorption (TPD) Fig. A2 – Schematic representation of the main chemical features in a graphene sheet, whit its typical surface functionalities (Liu, 2008) Table A1 - Desorption temperature of oxygen functional groups in carbon materials Oxygen functional groups Desorption products Desorption temperature (ºC) Carboxylic acid CO2 3502, 100-3003, 200-2504, Anhydride CO2+CO 6275, 5503, 250-5004, Lactone CO2 6275, 6603, 350-4004, Phenol CO 6303, 600-7004 Ether CO 6005 Quinone and half quinone 8003, 800-9004, 600-9506 1 (Aso et al., 2004); 2 (Otake and Jenkins, 1993) 3 (Figueiredo et al., 1999) 4 (Zielke et al., 1996) 5 (Zhuang et al., 1994) 6 (Marchon et al., 1988) Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 80 1.1.3 Fourier transform infrared (FTIR) 99,3 99,4 99,5 99,6 99,7 99,8 99,9 100 65085010501250145016501850 Transmitance (%) Wavenumber (cm-1) MWCNT MWCNT-Peroxidase 99,1 99,2 99,3 99,4 99,5 99,6 99,7 99,8 99,9 100 65085010501250145016501850 Transmitance (%) Wavenumber (cm-1) MWCNTox MWCNTox-Peroxidase Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 81 Fig. A3 – FTIR-ATR spectra of MWCNT, MWCNTox, MWCNTox-400 and MWCNTox-900, before (dash line) and after peroxidase immobilization (solid line) 99,4 99,5 99,6 99,7 99,8 99,9 100 65085010501250145016501850 Transmitance (%) Wavenumber (cm-1) MWCNTox-400 MWCNTox-400-Peroxidase 99,55 99,6 99,65 99,7 99,75 99,8 99,85 99,9 99,95 100 65085010501250145016501850 Transmitance (%) Wavenumber (cm-1) MWCNTox-900 MWCNTox-900-Peroxidase Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 82 1.1.4 Scanning electron microscope (SEM) Fig. A4 – SEM images of peroxidase adsorbed in MWCNTox-900 on different resolution. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 83 1.1.5 X-ray diffraction (XDR) Fig. A5 shows the X-ray diffraction (XRD) analysis of Fe3O4 and MWCNTs filled with Fe3O4. The composition of both materials and the dimension of the respective particles are also presented in Table A2. Fig. A5 - XRD diffractogram of magnetite (Fe3O4) (Fawal et al.) and CNT-Fe3O4 hybrid materials. Table A2 - Characterization of Fe3O4 and CNT-Fe3O4 hybrid material. Material Composition (% vol.) Particle dimention (nm) Magnetite Fe3O4 100% 14,54 ± 0,5 mMWCNTs Graphite 97% Fe3O4 3% 7,8 ± 3 18,9 ± 4 0 100 200 300 400 500 600 700 800 900 10 20 30 40 50 60 70 Intensity (a.u.) 2θo Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 84 Fig. A6 – mMWCNT attracted by a simple magnet Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 85 1.1.6 Transmission electron microscopy (TEM) TEM images of Fe3O4 nanoparticles and MWCNTs filled with Fe3O4 nanoparticles are shown in Fig. A7. Fig. A7 - TEM images of Fe3O4 (a) and mMWCNTs (b, c and d). (a) (b) (c) (d) 50 nm 250 nm 100 nm 100 nm Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 92 Table A6 - Comparison of Michaelis-Menten parameters obtained for immobilized peroxidase Michaelis-Menten CurveExpert Lineweaver-Burk (mM.min-1) 2,303±0,137 2,824 (mM) 0,064±0,018 0,135 Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 93 1.5 Storage and operational stability of immobilized peroxidase The reusability of MWCNTox-400-peroxidase conjugates was also determined. This procedure was identical to that of magnetic carbon nanotubes. The only difference was that this conjugates was separated from the reaction solution by filtration and then resuspended in a fresh ABTS solution and the process was repeated. The results are shown in Fig. A14. Fig. A14 – Reusability of peroxidase immobilized on MWCNTox-400 Fig. A15 – Reusability of peroxidase immobilized on a MWCNTs membrane 0 20 40 60 80 100 1 2 3 4 5 6 7 8 Peroxidase activity (%) Cycles 0 20 40 60 80 100 1 2 3 4 5 Peroxidase Activity (%) Cycles Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 94 1.6 Discoloration of dyes Table A7 – Properties of Reactive Black 5 Parameter Value C.I. Name Remazol Black 5 Commercial Name Reactive Black 5 Functional group Azo CAS Number 17095-24-8 EC Number 241-164-5 Mol. Mass (g.mol-1) 991,82 Mol. Formula C26H21N5Na4O19S6 Chemical Formula λmax 595 nm Purity (%) 55 Solubility 200 g.L-1 in water at 80 ºC Manufacturer Sigma-Aldrich Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 95 Table A8 - Properties of Reactive Blue 4 Parameter Value C.I. Name Procion® blue MX-R Commercial Name Reactive Blue 4 Functional group Amine CAS Number 13324-20-4 EC Number 236-363-9 Mol. Mass (g.mol-1) 637,43 Mol. Formula C23H14Cl2N6O8S2 Chemical Formula λmax 595 nm Purity (%) 35 Manufacturer Sigma-Aldrich Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 96 Table A9 - Properties of Bromophenol Blue Parameter Value C.I. Name Bromophenol Blue Commercial Name Bromophenol Blue Functional group Sulphonyl CAS Number 115-39-9 EC Number 204-086-2 Mol. Mass (g.mol-1) 669,96 Mol. Formula C19H10Br4O5S Chemical Formula λmax 595 nm Manufacturer Sigma-Aldrich Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 97 1.7 Bradford protein assay The protein concentration was determined using the Bradford protein assay. This method, a chromogenic assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue R250 (C43H44N3O7S2Na), that when bound to the protein, the red form of the dye turns blue. The red form of Coomassie dye causes a disruption of the protein’s native state exposing its hydrophobic regions. These regions bind via van der Waals forces to the non-polar region of the dye, causing a shift in the absorption maximum of the dye. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the concentration of protein present in the sample. In this way, a standard curve (Fig. A16) can be obtained using known protein concentrations. The obtained equation can be used to determine the protein concentration of other samples (proteins). There are other alternative methods, such as, Lowry and BCA protein assays. 1.7.1 Calibration curve The standard curves were obtained for a concentration range of 0.01-0.1 mg.L-1 and 0.1-1 mg.L-1 of a BSA solution. These results were used to obtain the peroxidase concentration in each sample. The standards were prepared mixing 1.5 mL of Coomassie Brilliant Blue with 50 μL of the protein solution, during 5 min. After this period, the absorbance was recorded by the spectrophotometer at 595 nm. The standard curves (absorbance vs protein concentration) are represented in Fig. A16. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 98 Fig. A16 – Standard curves for Bradford protein assay y = 0,5271x + 0,0202 R² = 0,993 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0 0,02 0,04 0,06 0,08 0,1 0,12 OD 595 BSA (mg.L-1) y = 0,689x - 0,0236 R² = 0,999 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 0,2 0,4 0,6 0,8 1 1,2 OD 595 BSA (mg.L-1) Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 99 Table A10 – Protein quantification for the immobilization process Sample Abs Concentration (mg.mL-1) Initial 0,0488 0,054 W1.A 0,0336 0,025 W1.B 0,0334 0,025 W2.A 0,0310 0,020 10.A 0,0325 0,023 10.B 0,0346 0,027 30.A 0,0211 0,002 30.B 0,0344 0,027 W1- supernatant of the first wash W2 - supernatant of the second wash 10. - supernatant of the sample after incubation at 50 ºC during 10 min 30. - supernatant of the sample after incubation at 50 ºC during 30 min Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 100 1.8 Isoelectric point The isoelectric point, or pI, is the pH at which the overall net charge on the protein is zero. It serves as a characteristic for every protein; proteins can be precipitated most easily at the pI, and similarly, charge interactions can be assessed when both the pI and pH of interest are known (Bommarius and Riebel-Bommarius, 2007). Amino acids in solution at neutral pH exist predominantly as dipolar ions called zwitterions. In this dipolar form, the amino group is protonated (-NH3+) and the carboxyl group is deprotonated (-COO-). The pI is determined by forming a pH gradient when an electric field is applied across a solution containing the protein. At this pH value, its electrophoretic mobility is also zero. When placed on an electrical field in a pH gradient, a protein will migrate until it reaches a position in the gel at which the pH is equal to the pI of the protein (Bommarius and Riebel-Bommarius, 2007). Fig. A17 – Isoelectric point focusing. Lac (Laccase), Stdr. (Standard) and Perox (Peroxidase), pH 4-6. Lac. Perox. Stdr. Stdr. Immobilization of Peroxidase on Functionalized Carbon Nanotubes for Synthesis of Biocatalysts with High Performance Appendix 101 1.9 Gel Electrophoresis SDS-Page Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE was performed in order to evaluate the purity of commercial peroxidase solution (Fig. A18). It was used an Amersham ECL Gel Box and an Amersham ECL Gel. The following instructions contain a short protocol describing the procedure adopted. 1.9.1 Preparations before a run Step Action 1 Prepare 1X running buffer by diluting 19 mL of Amersham ECL Gel Running Buffer, 10X in 171 mL water. 190 mL buffer is sufficient for one electrophoresis gel 2 Add 90 ml of 1× running buffer to each tank of Amersham ECL Gel Box 3 Cur open the gel package and gently remove Amersham ECL Gel from the package 4 Rinse the gel cassette with distilled water. Peel off the tapes from the two legs of the cassette. 5 Place Amersham ECL Gel in Amersham ECL Gel Box so that the well side of the cassette faces toward the cathode - and the other cassette leg faces toward the anode + 6 Place the safety lid on top of Amersham ECL Gel Box 7 Connect Amersham ECL Gel Box to the power supply EPS 301 and pre-run the gel for 12 minutes in 160 V 8 Once the pre-run is finished, switch off the power 9 Remove the safety lid 10 Wiggle the comb back and forth, and bring it straight up from the cassette to make the wells available for sample loading 11 Add 6 mL of 1X running buffer to the well container 1.9.2 Sample loading Step Action 1 Prepare the samples by adding sample and 2X sample buffer in a 1:1 mixture 2 Heat the samples at 95ºC for 5 minutes 3 Spin down the samples quickly in a microcentrifuge and load samples directly into the wells in the gel