- Email: [email protected]
In-situ immobilization of enzymes in mesoporous silicas
Solid State Sciences 13 (2011) 691e697
Contents lists available at ScienceDirect
Solid State Sciences journal homepage: www.elsevier.com/locate/ssscie
In-situ immobilization of enzymes in mesoporous silicas Esther Santalla a, Elías Serra a, Alvaro Mayoral b, José Losada c, Rosa M. Blanco a, Isabel Díaz a, * a
Instituto de Catálisis y Petroleoquímica, CSIC, c/Marie Curie 2, Cantoblanco, 28049 Madrid, Spain Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain c Escuela Técnica Superior de Ingenieros Industriales, Universidad Politécnica de Madrid, 28006 Madrid, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 January 2010 Received in revised form 14 September 2010 Accepted 15 September 2010 Available online 16 October 2010
Lipase from Candida antarctica B, horseradish peroxidase and laccase have been entrapped in silica cages rising mesoporous structures. Lipase and laccase yielded the highest structured mesoporous material whereas horseradish peroxidase may have altered the symmetry giving as a result mesocelullar foam (MCF) type of cages. The possible effect in the final structure of the material of the nature, size and surface structure of the proteins as well as the presence of various additives in the enzyme extracts is currently under investigations. Ó 2010 Elsevier Masson SAS. All rights reserved.
Dedicated to Prof. Osamu Terasaki on his retirement from Stockholm University. Keywords: Ordered mesoporous materials Enzyme immobilization Enzyme entrapment
1. Introduction Immobilization of homogeneous biocatalysts by encapsulation or entrapment methods lead to a nano-bioreactor in which enzyme molecules would be entrapped in isolated pores connected by smaller entrances. The sol-gel entrapment of enzymes is based on the condensation of the silica precursors around the enzyme molecules, so that a porous network is formed in which the protein is randomly entrapped. However, this technique yields a disordered structure of pores having a narrow size. On the other hand, silica Ordered Mesoporous Materials (OMM) offer tunable control of the porous network (including structural and textural properties) by simply adding a surfactant and adjusting the synthesis conditions, leading to a precise location of the biomolecules. The entrapment method (referred as in-situ immobilization) has not been widely used for the immobilization of enzymes on OMMs mainly because of the harsh synthesis conditions, pH and temperature, that may lead to enzyme denaturalization. However, recent developments in OMM field shows promising mild synthesis routes that yielded in few interesting attempts for enzyme entrapment. A highly ordered structure depends on many variables to be achieved, but the application of this technique may allow at least to obtain a controlled pore distribution and also to successfully remove the * Corresponding author. E-mail address: [email protected] (I. Díaz). 1293-2558/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.solidstatesciences.2010.09.015
surfactant. Fluorinated surfactants at neutral pH have succeeded to yield ordered hexagonal mesoporous silica to encapsulate glucose oxidase [1], and the method has been improved by using mixtures of various amphyphilics for one step entrapment of lipase [2]. Reports dealing with sol-gel synthesis [3] or xerogels [4] have also been tested, yielding barely structured solids (of that MCF type) and low enzyme loadings. In contrast with the precedent literature, our synthesis has been performed with triblock co-polymers (Pluronics), the most common type of templating agents nowadays, without the use of any additional chemicals and under mild conditions [5]. We have recently reported how cage-like mesoporous networks (such as SBA-16 and FDU-12) are able to reduce to a minimum desorption of lipase from Candida antarctica B [6]. In our recent investigations, we apply sol-gel encapsulation methods combined with the synthesis of an ordered porous network by simply adding a surfactant and adjusting the synthesis conditions. In the final material, the silica is built around the protein leading to a precise location of the biomolecules in silica cages whose entrances are smaller than the enzyme itself. Therefore, reagents and products may pass through, while maintaining the enzyme entrapped and ready for reuse [7]. The conditions in which the syntheses of the ordered mesoporous materials are performed had to be strongly modified. A pH of 3.5 was set for our experiments, still low but far from the typically values (0 < pH < 1). We also had to dispense with the hydrothermal
692
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
Fig. 1. Characterization results of the mesoporous solid obtained from CaLB. a) X ray diffraction pattern. b) high resolution SEM micrograph. c) TEM micrograph along [110] projection. d) HAADF image of another crystal recorded along the [110] orientation, the FFT diffractogram is shown inset.
treatment at high temperature that usually follows the initial reaction step. To compensate the mildness of the conditions, big concentrations of inorganic salts were added in some of the experiments, as they are known to promote the interaction between the surfactant micelles and the silica precursors [8]. Following this approach, we have used 3 enzymes with different surface properties in order to study the effect of the interactions enzyme e surfactant e silica on the synthesis of the siliceous mesoporous materials. These enzymes are: lipase from Candida antarctica B (CalB), having a hydrophobic surface, horseradish peroxidase (HRP) having a hydrophilic surface and isoelectric point (pI) 7.2, and laccase (PPO) also hydrophilic and with pI of 4.2. The possible effect of these parameters in the final structure of the material of the nature, size and surface structure of the proteins is envisaged in this manuscript. 2. Experimental section
Acetone was from Scharlau. The Substrates 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonate) ABTS and p-Nitrophenyl Acetate (p-NPA) were from Sigma (USA) and all the water was MiliQ. 2.2. In-situ immobilization In a typical synthesis 0.98 g of pluronic F127 surfactant were dissolved in 71 ml of water and the pH adjusted to 3.5 with HCl. KCl was added up to a final concentration of 0.5 M. 24.5 ml of enzyme solution were added, and the mixture was left to equilibrate for one hour. 3.75 g of silica source (TMOS) was added, and the mixture kept under stirring at 27 C for thirteen days. Samples of supernatant were withdrawn at certain times to monitorize the incorporation of the enzyme in the solid structure. This was performed by checking the protein content of the supernatants by the Bradford method [9]. Catalytic activity of the suspension was also assayed at different times. Finally the mixture was filtered, washed with a specific buffer and dried with acetone.
2.1. Chemicals 2.3. Removal of surfactant from the biocatalysts The enzymes extracts of HRP, PPO and CaLB were kindly donated by Novozymes (Spain) and the isolated enzymes peroxidase (HRP*) from horseradish Type VI-A and tyrosinase (PPO*) from mushroom were purchased from Sigma (USA). Pluronic F127 was commercially available from BASF. The silica source Tetramethoxysilane (TMOS) was from Lancaster. KCl, HCl, NaOH and H2O2 were from Panreac.
Among the different treatments, the most efficient method for the surfactant removal was a solution of HCl in water at pH ¼ 3.5 and refluxed at 38 C. Typically, 1 g of OMM was dispersed in 150 ml of HCl solution for 24 h with one change of solvent after 6 h. The solid was filtered and dried with acetone between subsequent
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
changes. Thermogravimetrical analysis of the samples was performed in order to check quantitative removal of the surfactant (data not shown). The presence of enzyme in the OMM and the protein leaching were checked by Bradford method and electrophoresis of all the preparations before and after the extraction of the surfactant. 2.4. Characterization All the samples were extensively characterized with a combination of several techniques. X ray diffraction (XRD) was performed in a Seifert XRD 3000 P diffractometer operating at low angle. Transmission electron microscopy (TEM) was performed using a Philips Tecnai G2, 200 kV, TEM equipped with Schottky-type field emission gun, ultra-high resolution pole piece (Cs ¼ 0.5 mm) operating at 200 kV. High resolution scanning electron microscopy (SEM) was performed in a FEG Hitachi S- 5500 ultra-high resolution electron microscope (0.4 nm at 30 kV) with BF/DF Duo-STEM detector. For TEM observation the powders were deeply crushed using a mortar and pestle, dispersed in ethanol and dropped on holey copper carbon grids. For SEM analysis, the materials were dispersed in ethanol and dropped onto a holey carbon copper microgrid. In order to observe the original morphology the samples, in this case, were not crushed. Conventional SEM micrographs were collected with a JEOL JSM6400 Philips XL30 operating at 20 kV. Nitrogen adsorption/desorption isotherms were carried out in a Micromeritics TriStar3000 apparatus following the BET procedure. Thermogravimetrical analyses (TGA) were recorded on a Perking-Elmer TGA7 equipment, scanning a temperature range of 20e900 C at a heating rate of 10 C/min. 2.5. Enzyme activity
693
3. Results and discussion When CaLB was used in the synthesis gel, the resulting X ray diffraction pattern (Fig. 1a) of the sample is in good agreement whit the cage-like cubic Fm-3 m structure, as it has been already described [7]. The analysis of the samples by SEM reveals irregular morphology with particles sizes ranging from 2 to 8 mm. On the other hand, high resolution SEM Fig. 1b was carried out on noncoated sample allowing observing the pore openings up to the surface of the particles. To corroborate the high degree of structural order, TEM analyses of the samples was carried out. The corresponding Fm-3 m space group could be indexed thanks to the application of tilting series to obtain electron diffraction patterns along the different axes. As an example, Fig. 1c shows a typical image of a [110] projection in which some intergrowths could be observed [11]. High resolution images were also recorded in scanning transmission electron microscopy mode (STEM) with a high angle annular dark field detector (HAADF), Fig. 1d. In this mode the beam is converged and scanned over the material and only electrons which are scattered at high angle are collected to form the image, therefore the contrast obtained in this type of images is strongly related to the atomic number of the element. Fig. 1d presents a well defined porous solid with stacking faults in the structure, which was confirmed by the diffuse spots obtained in the FFT diffractogram. For the textural characterization of the silica matrix, it is required to remove both enzyme and surfactant molecules from inside the pores which is usually carried out by calcination in air at 550 C. The mesoporous structure was corroborated by XRD after removal of the organic molecules inside the pores by calcination. N2 adsorption/desorption isotherm is plotted in Fig. 2. It shows the sharp step at a relative pressure of around 4 mm Hg, typical of
The qualitative protein analysis of the enzymes, analysis postsynthesis and post- surfactant removal were determined through electrophoresis [10]. Quantitative protein content of all the enzyme solutions was measured using Bradford method [9]. Catalytic activity assays were carried out by following spectrophotometrically the transformation of substrates (p-NPA at 348 nm for lipase activity and ABTS at 436 and 414 nm for PPO and HRP activity respectively). An Agilent 8453 UV-Vis spectrophotometer equipped with stirring device and temperature controller was used for these assays. Further catalytic details will be reported in a more specific manuscript. 2.6. Electrochemical measurements Electrochemical measurements were performed using an Ecochemie Autolab PGSTAT 12. All experiments were carried out at 20e21 C in a conventional three-electrode cell, consisting of a saturated Calomel reference electrode (SCE), a Pt wire as auxiliary electrode and a modified carbon paste electrode as working electrode. The electrochemical measurements were performed in 0.1 M phosphate buffer with 0.1 M NaClO4 (pH 7.0). All solutions were deoxygenated by bubbling high-purity nitrogen for at least 15 min. 2.7. Preparation of carbon paste electrode The electrodes were constructed by mixing 25 mg of mesoporous silica material with immobilized enzyme and 25 mg of graphite powder. Paraffin oil was added and mixed vigorously until get a homogeneous paste. The paste was placed in a hole (3.0 mm diameter, 4 mm deep at the end of a carbon paste electrode (BAS MF-2010) (A ¼ 0.07 cm2)). The electrodes were polished with a piece of weighing paper.
Fig. 2. N2 adsorption/desorption isotherms of the materials obtained in the presence of CaLB, PPO, PPO*, HRP, and HRP*.
694
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
mesoporous solids, along with the hysteresis cycle defined by the IUPAC as H2 closely associated with cage-like pores. Regarding the loading of the enzyme inside the silica matrix, the presence of enzyme after surfactant removal treatments was corroborated in all the cases by electrophoresis. In a final study, the Bradford method show the partial incorporation of the enzyme inside the material accompanied with a fair catalytic efficiency [7]. When using PPO in the synthesis of the mesoporous materials, once again XRD profiles (Fig. 3a) and TEM micrographs (Fig. 3b) show a cage-like cubic material Fm-3 m space group, with the absence of intergrowth in this case. SEM analysis (Fig. 3c) reveals different polyhedral particles and globular morphology characteristic of these kinds of cage-like solids. These particles, that are 3e6 mm large, are further aggregated in different degrees. High resolution SEM shows again the pores openings up to the surface. The structure remains stable after calcination. Fig. 2 shows the N2 adsorption/desorption isotherm similar to that of the material prepared in the presence of CalB. However, in this case, Bradford and electrophoresis methods showed no incorporation of the enzyme in
the solid material, meaning that the enzyme did not interact with the surfactant molecules prior to the condensation of the silica. The synthesis was also tested with lyophilized PPO (PPO*) under the same conditions however, no improvement of the enzyme entrapment was achieved yielding large agglomerates of highly packed small nanoparticles instead of the single large globular particles previously described (Fig. 4). Besides, the N2 adsorption/ desorption isotherms (Fig. 2) do not show a sharp step usually observed for mesoporous materials. Finally, when horseradish peroxidase (HRP) is present in the synthesis gel, the broad but intense band at low angle in the XRD pattern primarily indicates a disordered mesoporous materials obtained both in the presence of HRP crude and isolated HRP* (Fig. 5a). The analyses of the samples by SEM reveal a material formed by large agglomerates of very small nanoparticles in both cases (Fig. 5b). However, only mesopores could be observed by TEM (Fig. 5c) in the HRP synthesis, resulting in mesocelullar foam (MCF) type of cages which implies a disordered structure and therefore explains the broad and less intense low angle XRD peak.
Fig. 3. Characterization results of the mesoporous solid obtained from PPO. a) X ray diffraction pattern, b) TEM micrograph along [110] projection, and c) conventional and high resolution SEM micrographs.
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
695
Fig. 4. SEM micrographs of the amorphous materials obtained in the presence of PPO*.
On the other hand, the N2 adsorption/desorption isotherms in Fig. 2 shows a narrow pore size distribution of mesopores in the HRP sample, but mayor macroporosity or porosity due to pores between particles in the HRP* sample (Fig. 2). Thus, the low angle broad peak observed in the XRD fro HRP* pattern is related to the small size of the nanoparticles observed by SEM rather than to a disorder mesoporous structure. Bradford method shows the total incorporation of the enzyme on the HRP material. Catalytic test also shows that the incorporation is efficient and prevails after surfactant removal. Whereas, when the synthesis was carried out in the presence of isolated HRP*, no incorporation of the enzyme in the material is observed. According to the above detailed results, CaLB is incorporated and is highly efficient to obtain an ordered mesoporous structure with an active enzyme in the final material. However, ordered structure was not obtained with HRP, and not even entrapped biomolecules within in the siliceous skeleton could be detected with PPO. CaLB has the unique feature to display a hydrophobic external area which probably interacts with the surfactant contributing to the formation of micelles. Thus, the fact that HRP or PPO failed to drive the formation of ordered structures with enzyme entrapped might be related to the absence of this hydrophobic surface that allows interaction with the surfactant in an efficient manner. Both enzymes are highly glycosilated (up to 20% of glycoside content), therefore their respective external surfaces are polar and this might explain that the right organization of the micelles could be altered. On the other hand, syntheses of the biocatalysts were performed at pH 3.5, close to the pI of PPO (4.2), and far from pI of lipase (6) and HRP (7.2). That means that at the pH of work lipase is positively charged and electrostatic interactions should drive the process. But the highly hydrophobic domain of the enzyme makes hydrophobic interactions to prevail on electrostatic ones and the ordered structure can be constructed. HRP has a higher pI, so it is strongly charged therefore the driving forces of the process are electrostatic interactions. Yet, the scenario seems to be stable enough to allow for some interaction with the surfactant molecules, although the enzyme may be altering noticeably the packing and further growth of the mesoporous material (SEM analysis showed agglomerated nanoparticles, and TEM showed mesocelullar foam). For these later samples, an electrochemical study was carried out on order to evaluate the stability of the entrapped HRP. For this proposes, the in situ HRP-Mesoporous material has been compared with an HRP supported on a conventional SBA-15 post-synthesis. The behaviour of the HRP immobilized on SBA-15 was similar to that previously observed with other types of compounds on MCM41, a similar material to the SBA-15 [12].
Fig. 5. a) XRD of the material obtained in the presence of HRP and HRP*; b) SEM micrographs of the amorphous materials obtained in the presence of HRP*; c)TEM micrograph of the MCF obtained in the presence of HRP.
Phenols can be detected employing HRP in the presence of hydrogen peroxide. The enzymatic mechanism involved is the enzyme oxidation by hydrogen peroxide followed of reduction by phenol. This last reaction converts phenol into quinone and/or free radicals, which are in turn electroactive and are reduced at the electrode surface regenerating the phenol groups. The HRP
696
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
analytical signal is therefore the electrochemical regeneration of phenol oxidized by the enzyme. In any case, the peroxidases are able to experiment direct electron transfer between the enzyme and the electrode without mediators. Usually this reaction is a slow process and an electron mediator is used to improve the rate of the electron transfer. Horseradish peroxidase catalyzes the reduction of H2O2, however the immobilization of HRP blocks the direct electron transfer between the enzyme and the electrode, probably due to the insulator characteristics of the matrix where the enzyme is encapsulated. In this case HRP in the presence of hydrogen peroxide can only be regenerated by electrochemical reduction through a mediator as phenol previously reduced on the electrode. This behaviour allows increasing the sensitivity of the biosensor for phenolic compounds [13] and can be verified by linear sweep voltammetry (Fig. 6). The results presented here compare the effect of the in-situ immobilization process (HRP-mesoporous) with a more conventional post grafting method. The electrode built with enzyme postgrafted on a mesoporous material called HRP/SBA-15 produces a different electrochemical response that than obtained with electrodes constructed with the in situ HRP-Mesoporous material. As can be seen in the Fig. 6A using the electrodes prepared with HRP-Mesoporous material, in which the HRP has been immobilized
during synthesis of the mesoporous material (HRP in situ), only one type of response is observed that corresponds to the addition of mediator. In the presence of H2O2 the blocking effect avoids to a great extent the direct reduction of HRP (line b). The addition of phenol, which acts a mediator, causes an appreciable increase of the current in all potentials range swept (line c). The presence of a mediator allows the enzyme reduction by phenol that has been regenerated on the electrode giving rise to an electrocatalytical process. On the contrary when the electrodes are constructed with HRP/ SBA-15 (Fig. 6B) the presence of H2O2 produces an increase of the current in all potential range (line b). Furthermore when phenol is added to this dissolution a higher reduction current can be detected (line c). This behaviour can be explained assuming that two types of enzyme are supported on the SBA-15. Some HRP molecules will be anchored to the pore openings or even leached from the silica matrix and somehow they remain entrapped independently in the carbon paste. These HRP molecules are in direct contact with the electrode allowing the direct electron transfer from the electrode. And other type of HRP is presented in the material which would be confined in the interior of the SBA-15 channels being totally isolated electrochemically by the silica matrix. This confined HRP can be catalytically active giving an electrochemical response when a mediator is added.
Fig. 6. Linear sweep voltammogram obtained for (A) mesoporous-HRP- and (B) HRP/SBA-15- modified carbon paste electrodes (line a) in 0.1 M phosphate buffer solution, pH 7.0, (line b) in the presence of 35 mmol1 hydrogen peroxide, (line c) 35 mmol1 hydrogen peroxide and 100 mmol1 of phenol. Potential scan rate 10 mV s1. (C) Scheme of in situ HRPMesoporous material in which all the enzyme molecules are isolated in ideal meso-cages. (D) Scheme of the scenario in HRP/SBA-15 electrode, in which two types of HRP molecules are presented: some inside the channels and some outside the channels.
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
The results obtained show that the immobilization process in the case of HRP post-grafted on SBA-15 is efficient, but not very selective in terms of confinement. On other hand the in-situ immobilization of HRP in mesoporous materials yields enzyme molecules that are successfully isolated by the silica, i.e., encapsulated in isolated cages in meoporous material as represented in the scheme (Fig. 6B). Therefore, we can conclude that the encapsulation method using surfactant to assist the synthesis of a mesoporous material has worked successfully since enzyme keeps “alive” and in this case also it stays confined selectively inside mesoporous cages. 4. Conclusions From the results observed and discussed we can conclude that the parameters influencing the in situ synthesis of ordered mesoporous biocatalysts are the following: Hydrophobicity of the surface of the protein: If the protein is hydrophobic enough the siliceous material is synthesized even in the presence of electrostatic charges. When the protein is charged, the relevance of this depends on the protein surface. If the surface is hydrophobic enough, the hydrophobic interactions prevail and the right structure is formed. If the surface is hydrophilic then the charges may be the driving force of the process, and no order is obtained in the final material. The surfactant cannot form the micelles properly and even nonporous nanostructures can be formed. The relevance of these results relies on the fact that they help us to understand the complex process and to envisage parameters that
697
should be studied in order to reach a methodology enabling to establish a general protocol to get the synthesis of OMM using any enzyme.
Acknowledgements The authors acknowledge the Spanish Ministry of Education and Science for the financial support (MAT-2006-04107).
References [1] J.L. Blin, C. Gérardin, C. Carteret, L. Rodehüser, C. Selve, M.J. Stébé, Chem. Mater. 17 (2005) 1479. [2] M. Mureseanu, A. Galarneau, G. Renard, F. Fajula, Langmu 21 (2005) 4648. [3] Y. Wei, H. Dong, J. Xu, Q. Feng Chem, Phys. Chem. 9 (2002) 802. [4] X.Y. Yang, Z.Q. Li, B. Liu, A. Klein-Hofmann, G. Tian, Y.F. Feng, et al., Adv. Mater. 18 (2006) 410. [5] E. Serra, V. Alfredsson, R.M. Blanco, I. Díaz, Stud. Surf. Sci. Catal. 174 (2008) 369. [6] E. Serra, A. Mayoral, Y. Sakamoto, R.M. Blanco, I. Díaz, Micropor. Mesopor. Mater. 114 (2008) 201e213. [7] E. Serra, R.M. Blanco, V. Alfredsson, I. Díaz, Micropor. Mesopor. Mater. 129 (2010) 173e178. [8] Y. Wan, D.Y. Zhao, Chem. Rev. 107 (2007) 2821. [9] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [10] B.J. Davis, N.Y. Ann, Acad. Sci. 121 (1964) 404. [11] Y. Sakamoto, I. Díaz, O. Terasaki, D. Zhao, J. Pérez-Pariente, J.M. Kim, et al., Chem. B. 106 (2002) 3118. [12] I. Díaz, B. García, B. Alonso, C.M. Casado, M. Morán, J. Losada, et al., Chem. Mater. 15 (2003) 1073. [13] S.S. Rosatto, L.T. Kubota, G. de Oliveira Neto, Anal. Chim. Acta 390 (1999) 65.
Contents lists available at ScienceDirect
Solid State Sciences journal homepage: www.elsevier.com/locate/ssscie
In-situ immobilization of enzymes in mesoporous silicas Esther Santalla a, Elías Serra a, Alvaro Mayoral b, José Losada c, Rosa M. Blanco a, Isabel Díaz a, * a
Instituto de Catálisis y Petroleoquímica, CSIC, c/Marie Curie 2, Cantoblanco, 28049 Madrid, Spain Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain c Escuela Técnica Superior de Ingenieros Industriales, Universidad Politécnica de Madrid, 28006 Madrid, Spain b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 January 2010 Received in revised form 14 September 2010 Accepted 15 September 2010 Available online 16 October 2010
Lipase from Candida antarctica B, horseradish peroxidase and laccase have been entrapped in silica cages rising mesoporous structures. Lipase and laccase yielded the highest structured mesoporous material whereas horseradish peroxidase may have altered the symmetry giving as a result mesocelullar foam (MCF) type of cages. The possible effect in the final structure of the material of the nature, size and surface structure of the proteins as well as the presence of various additives in the enzyme extracts is currently under investigations. Ó 2010 Elsevier Masson SAS. All rights reserved.
Dedicated to Prof. Osamu Terasaki on his retirement from Stockholm University. Keywords: Ordered mesoporous materials Enzyme immobilization Enzyme entrapment
1. Introduction Immobilization of homogeneous biocatalysts by encapsulation or entrapment methods lead to a nano-bioreactor in which enzyme molecules would be entrapped in isolated pores connected by smaller entrances. The sol-gel entrapment of enzymes is based on the condensation of the silica precursors around the enzyme molecules, so that a porous network is formed in which the protein is randomly entrapped. However, this technique yields a disordered structure of pores having a narrow size. On the other hand, silica Ordered Mesoporous Materials (OMM) offer tunable control of the porous network (including structural and textural properties) by simply adding a surfactant and adjusting the synthesis conditions, leading to a precise location of the biomolecules. The entrapment method (referred as in-situ immobilization) has not been widely used for the immobilization of enzymes on OMMs mainly because of the harsh synthesis conditions, pH and temperature, that may lead to enzyme denaturalization. However, recent developments in OMM field shows promising mild synthesis routes that yielded in few interesting attempts for enzyme entrapment. A highly ordered structure depends on many variables to be achieved, but the application of this technique may allow at least to obtain a controlled pore distribution and also to successfully remove the * Corresponding author. E-mail address: [email protected] (I. Díaz). 1293-2558/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.solidstatesciences.2010.09.015
surfactant. Fluorinated surfactants at neutral pH have succeeded to yield ordered hexagonal mesoporous silica to encapsulate glucose oxidase [1], and the method has been improved by using mixtures of various amphyphilics for one step entrapment of lipase [2]. Reports dealing with sol-gel synthesis [3] or xerogels [4] have also been tested, yielding barely structured solids (of that MCF type) and low enzyme loadings. In contrast with the precedent literature, our synthesis has been performed with triblock co-polymers (Pluronics), the most common type of templating agents nowadays, without the use of any additional chemicals and under mild conditions [5]. We have recently reported how cage-like mesoporous networks (such as SBA-16 and FDU-12) are able to reduce to a minimum desorption of lipase from Candida antarctica B [6]. In our recent investigations, we apply sol-gel encapsulation methods combined with the synthesis of an ordered porous network by simply adding a surfactant and adjusting the synthesis conditions. In the final material, the silica is built around the protein leading to a precise location of the biomolecules in silica cages whose entrances are smaller than the enzyme itself. Therefore, reagents and products may pass through, while maintaining the enzyme entrapped and ready for reuse [7]. The conditions in which the syntheses of the ordered mesoporous materials are performed had to be strongly modified. A pH of 3.5 was set for our experiments, still low but far from the typically values (0 < pH < 1). We also had to dispense with the hydrothermal
692
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
Fig. 1. Characterization results of the mesoporous solid obtained from CaLB. a) X ray diffraction pattern. b) high resolution SEM micrograph. c) TEM micrograph along [110] projection. d) HAADF image of another crystal recorded along the [110] orientation, the FFT diffractogram is shown inset.
treatment at high temperature that usually follows the initial reaction step. To compensate the mildness of the conditions, big concentrations of inorganic salts were added in some of the experiments, as they are known to promote the interaction between the surfactant micelles and the silica precursors [8]. Following this approach, we have used 3 enzymes with different surface properties in order to study the effect of the interactions enzyme e surfactant e silica on the synthesis of the siliceous mesoporous materials. These enzymes are: lipase from Candida antarctica B (CalB), having a hydrophobic surface, horseradish peroxidase (HRP) having a hydrophilic surface and isoelectric point (pI) 7.2, and laccase (PPO) also hydrophilic and with pI of 4.2. The possible effect of these parameters in the final structure of the material of the nature, size and surface structure of the proteins is envisaged in this manuscript. 2. Experimental section
Acetone was from Scharlau. The Substrates 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonate) ABTS and p-Nitrophenyl Acetate (p-NPA) were from Sigma (USA) and all the water was MiliQ. 2.2. In-situ immobilization In a typical synthesis 0.98 g of pluronic F127 surfactant were dissolved in 71 ml of water and the pH adjusted to 3.5 with HCl. KCl was added up to a final concentration of 0.5 M. 24.5 ml of enzyme solution were added, and the mixture was left to equilibrate for one hour. 3.75 g of silica source (TMOS) was added, and the mixture kept under stirring at 27 C for thirteen days. Samples of supernatant were withdrawn at certain times to monitorize the incorporation of the enzyme in the solid structure. This was performed by checking the protein content of the supernatants by the Bradford method [9]. Catalytic activity of the suspension was also assayed at different times. Finally the mixture was filtered, washed with a specific buffer and dried with acetone.
2.1. Chemicals 2.3. Removal of surfactant from the biocatalysts The enzymes extracts of HRP, PPO and CaLB were kindly donated by Novozymes (Spain) and the isolated enzymes peroxidase (HRP*) from horseradish Type VI-A and tyrosinase (PPO*) from mushroom were purchased from Sigma (USA). Pluronic F127 was commercially available from BASF. The silica source Tetramethoxysilane (TMOS) was from Lancaster. KCl, HCl, NaOH and H2O2 were from Panreac.
Among the different treatments, the most efficient method for the surfactant removal was a solution of HCl in water at pH ¼ 3.5 and refluxed at 38 C. Typically, 1 g of OMM was dispersed in 150 ml of HCl solution for 24 h with one change of solvent after 6 h. The solid was filtered and dried with acetone between subsequent
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
changes. Thermogravimetrical analysis of the samples was performed in order to check quantitative removal of the surfactant (data not shown). The presence of enzyme in the OMM and the protein leaching were checked by Bradford method and electrophoresis of all the preparations before and after the extraction of the surfactant. 2.4. Characterization All the samples were extensively characterized with a combination of several techniques. X ray diffraction (XRD) was performed in a Seifert XRD 3000 P diffractometer operating at low angle. Transmission electron microscopy (TEM) was performed using a Philips Tecnai G2, 200 kV, TEM equipped with Schottky-type field emission gun, ultra-high resolution pole piece (Cs ¼ 0.5 mm) operating at 200 kV. High resolution scanning electron microscopy (SEM) was performed in a FEG Hitachi S- 5500 ultra-high resolution electron microscope (0.4 nm at 30 kV) with BF/DF Duo-STEM detector. For TEM observation the powders were deeply crushed using a mortar and pestle, dispersed in ethanol and dropped on holey copper carbon grids. For SEM analysis, the materials were dispersed in ethanol and dropped onto a holey carbon copper microgrid. In order to observe the original morphology the samples, in this case, were not crushed. Conventional SEM micrographs were collected with a JEOL JSM6400 Philips XL30 operating at 20 kV. Nitrogen adsorption/desorption isotherms were carried out in a Micromeritics TriStar3000 apparatus following the BET procedure. Thermogravimetrical analyses (TGA) were recorded on a Perking-Elmer TGA7 equipment, scanning a temperature range of 20e900 C at a heating rate of 10 C/min. 2.5. Enzyme activity
693
3. Results and discussion When CaLB was used in the synthesis gel, the resulting X ray diffraction pattern (Fig. 1a) of the sample is in good agreement whit the cage-like cubic Fm-3 m structure, as it has been already described [7]. The analysis of the samples by SEM reveals irregular morphology with particles sizes ranging from 2 to 8 mm. On the other hand, high resolution SEM Fig. 1b was carried out on noncoated sample allowing observing the pore openings up to the surface of the particles. To corroborate the high degree of structural order, TEM analyses of the samples was carried out. The corresponding Fm-3 m space group could be indexed thanks to the application of tilting series to obtain electron diffraction patterns along the different axes. As an example, Fig. 1c shows a typical image of a [110] projection in which some intergrowths could be observed [11]. High resolution images were also recorded in scanning transmission electron microscopy mode (STEM) with a high angle annular dark field detector (HAADF), Fig. 1d. In this mode the beam is converged and scanned over the material and only electrons which are scattered at high angle are collected to form the image, therefore the contrast obtained in this type of images is strongly related to the atomic number of the element. Fig. 1d presents a well defined porous solid with stacking faults in the structure, which was confirmed by the diffuse spots obtained in the FFT diffractogram. For the textural characterization of the silica matrix, it is required to remove both enzyme and surfactant molecules from inside the pores which is usually carried out by calcination in air at 550 C. The mesoporous structure was corroborated by XRD after removal of the organic molecules inside the pores by calcination. N2 adsorption/desorption isotherm is plotted in Fig. 2. It shows the sharp step at a relative pressure of around 4 mm Hg, typical of
The qualitative protein analysis of the enzymes, analysis postsynthesis and post- surfactant removal were determined through electrophoresis [10]. Quantitative protein content of all the enzyme solutions was measured using Bradford method [9]. Catalytic activity assays were carried out by following spectrophotometrically the transformation of substrates (p-NPA at 348 nm for lipase activity and ABTS at 436 and 414 nm for PPO and HRP activity respectively). An Agilent 8453 UV-Vis spectrophotometer equipped with stirring device and temperature controller was used for these assays. Further catalytic details will be reported in a more specific manuscript. 2.6. Electrochemical measurements Electrochemical measurements were performed using an Ecochemie Autolab PGSTAT 12. All experiments were carried out at 20e21 C in a conventional three-electrode cell, consisting of a saturated Calomel reference electrode (SCE), a Pt wire as auxiliary electrode and a modified carbon paste electrode as working electrode. The electrochemical measurements were performed in 0.1 M phosphate buffer with 0.1 M NaClO4 (pH 7.0). All solutions were deoxygenated by bubbling high-purity nitrogen for at least 15 min. 2.7. Preparation of carbon paste electrode The electrodes were constructed by mixing 25 mg of mesoporous silica material with immobilized enzyme and 25 mg of graphite powder. Paraffin oil was added and mixed vigorously until get a homogeneous paste. The paste was placed in a hole (3.0 mm diameter, 4 mm deep at the end of a carbon paste electrode (BAS MF-2010) (A ¼ 0.07 cm2)). The electrodes were polished with a piece of weighing paper.
Fig. 2. N2 adsorption/desorption isotherms of the materials obtained in the presence of CaLB, PPO, PPO*, HRP, and HRP*.
694
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
mesoporous solids, along with the hysteresis cycle defined by the IUPAC as H2 closely associated with cage-like pores. Regarding the loading of the enzyme inside the silica matrix, the presence of enzyme after surfactant removal treatments was corroborated in all the cases by electrophoresis. In a final study, the Bradford method show the partial incorporation of the enzyme inside the material accompanied with a fair catalytic efficiency [7]. When using PPO in the synthesis of the mesoporous materials, once again XRD profiles (Fig. 3a) and TEM micrographs (Fig. 3b) show a cage-like cubic material Fm-3 m space group, with the absence of intergrowth in this case. SEM analysis (Fig. 3c) reveals different polyhedral particles and globular morphology characteristic of these kinds of cage-like solids. These particles, that are 3e6 mm large, are further aggregated in different degrees. High resolution SEM shows again the pores openings up to the surface. The structure remains stable after calcination. Fig. 2 shows the N2 adsorption/desorption isotherm similar to that of the material prepared in the presence of CalB. However, in this case, Bradford and electrophoresis methods showed no incorporation of the enzyme in
the solid material, meaning that the enzyme did not interact with the surfactant molecules prior to the condensation of the silica. The synthesis was also tested with lyophilized PPO (PPO*) under the same conditions however, no improvement of the enzyme entrapment was achieved yielding large agglomerates of highly packed small nanoparticles instead of the single large globular particles previously described (Fig. 4). Besides, the N2 adsorption/ desorption isotherms (Fig. 2) do not show a sharp step usually observed for mesoporous materials. Finally, when horseradish peroxidase (HRP) is present in the synthesis gel, the broad but intense band at low angle in the XRD pattern primarily indicates a disordered mesoporous materials obtained both in the presence of HRP crude and isolated HRP* (Fig. 5a). The analyses of the samples by SEM reveal a material formed by large agglomerates of very small nanoparticles in both cases (Fig. 5b). However, only mesopores could be observed by TEM (Fig. 5c) in the HRP synthesis, resulting in mesocelullar foam (MCF) type of cages which implies a disordered structure and therefore explains the broad and less intense low angle XRD peak.
Fig. 3. Characterization results of the mesoporous solid obtained from PPO. a) X ray diffraction pattern, b) TEM micrograph along [110] projection, and c) conventional and high resolution SEM micrographs.
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
695
Fig. 4. SEM micrographs of the amorphous materials obtained in the presence of PPO*.
On the other hand, the N2 adsorption/desorption isotherms in Fig. 2 shows a narrow pore size distribution of mesopores in the HRP sample, but mayor macroporosity or porosity due to pores between particles in the HRP* sample (Fig. 2). Thus, the low angle broad peak observed in the XRD fro HRP* pattern is related to the small size of the nanoparticles observed by SEM rather than to a disorder mesoporous structure. Bradford method shows the total incorporation of the enzyme on the HRP material. Catalytic test also shows that the incorporation is efficient and prevails after surfactant removal. Whereas, when the synthesis was carried out in the presence of isolated HRP*, no incorporation of the enzyme in the material is observed. According to the above detailed results, CaLB is incorporated and is highly efficient to obtain an ordered mesoporous structure with an active enzyme in the final material. However, ordered structure was not obtained with HRP, and not even entrapped biomolecules within in the siliceous skeleton could be detected with PPO. CaLB has the unique feature to display a hydrophobic external area which probably interacts with the surfactant contributing to the formation of micelles. Thus, the fact that HRP or PPO failed to drive the formation of ordered structures with enzyme entrapped might be related to the absence of this hydrophobic surface that allows interaction with the surfactant in an efficient manner. Both enzymes are highly glycosilated (up to 20% of glycoside content), therefore their respective external surfaces are polar and this might explain that the right organization of the micelles could be altered. On the other hand, syntheses of the biocatalysts were performed at pH 3.5, close to the pI of PPO (4.2), and far from pI of lipase (6) and HRP (7.2). That means that at the pH of work lipase is positively charged and electrostatic interactions should drive the process. But the highly hydrophobic domain of the enzyme makes hydrophobic interactions to prevail on electrostatic ones and the ordered structure can be constructed. HRP has a higher pI, so it is strongly charged therefore the driving forces of the process are electrostatic interactions. Yet, the scenario seems to be stable enough to allow for some interaction with the surfactant molecules, although the enzyme may be altering noticeably the packing and further growth of the mesoporous material (SEM analysis showed agglomerated nanoparticles, and TEM showed mesocelullar foam). For these later samples, an electrochemical study was carried out on order to evaluate the stability of the entrapped HRP. For this proposes, the in situ HRP-Mesoporous material has been compared with an HRP supported on a conventional SBA-15 post-synthesis. The behaviour of the HRP immobilized on SBA-15 was similar to that previously observed with other types of compounds on MCM41, a similar material to the SBA-15 [12].
Fig. 5. a) XRD of the material obtained in the presence of HRP and HRP*; b) SEM micrographs of the amorphous materials obtained in the presence of HRP*; c)TEM micrograph of the MCF obtained in the presence of HRP.
Phenols can be detected employing HRP in the presence of hydrogen peroxide. The enzymatic mechanism involved is the enzyme oxidation by hydrogen peroxide followed of reduction by phenol. This last reaction converts phenol into quinone and/or free radicals, which are in turn electroactive and are reduced at the electrode surface regenerating the phenol groups. The HRP
696
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
analytical signal is therefore the electrochemical regeneration of phenol oxidized by the enzyme. In any case, the peroxidases are able to experiment direct electron transfer between the enzyme and the electrode without mediators. Usually this reaction is a slow process and an electron mediator is used to improve the rate of the electron transfer. Horseradish peroxidase catalyzes the reduction of H2O2, however the immobilization of HRP blocks the direct electron transfer between the enzyme and the electrode, probably due to the insulator characteristics of the matrix where the enzyme is encapsulated. In this case HRP in the presence of hydrogen peroxide can only be regenerated by electrochemical reduction through a mediator as phenol previously reduced on the electrode. This behaviour allows increasing the sensitivity of the biosensor for phenolic compounds [13] and can be verified by linear sweep voltammetry (Fig. 6). The results presented here compare the effect of the in-situ immobilization process (HRP-mesoporous) with a more conventional post grafting method. The electrode built with enzyme postgrafted on a mesoporous material called HRP/SBA-15 produces a different electrochemical response that than obtained with electrodes constructed with the in situ HRP-Mesoporous material. As can be seen in the Fig. 6A using the electrodes prepared with HRP-Mesoporous material, in which the HRP has been immobilized
during synthesis of the mesoporous material (HRP in situ), only one type of response is observed that corresponds to the addition of mediator. In the presence of H2O2 the blocking effect avoids to a great extent the direct reduction of HRP (line b). The addition of phenol, which acts a mediator, causes an appreciable increase of the current in all potentials range swept (line c). The presence of a mediator allows the enzyme reduction by phenol that has been regenerated on the electrode giving rise to an electrocatalytical process. On the contrary when the electrodes are constructed with HRP/ SBA-15 (Fig. 6B) the presence of H2O2 produces an increase of the current in all potential range (line b). Furthermore when phenol is added to this dissolution a higher reduction current can be detected (line c). This behaviour can be explained assuming that two types of enzyme are supported on the SBA-15. Some HRP molecules will be anchored to the pore openings or even leached from the silica matrix and somehow they remain entrapped independently in the carbon paste. These HRP molecules are in direct contact with the electrode allowing the direct electron transfer from the electrode. And other type of HRP is presented in the material which would be confined in the interior of the SBA-15 channels being totally isolated electrochemically by the silica matrix. This confined HRP can be catalytically active giving an electrochemical response when a mediator is added.
Fig. 6. Linear sweep voltammogram obtained for (A) mesoporous-HRP- and (B) HRP/SBA-15- modified carbon paste electrodes (line a) in 0.1 M phosphate buffer solution, pH 7.0, (line b) in the presence of 35 mmol1 hydrogen peroxide, (line c) 35 mmol1 hydrogen peroxide and 100 mmol1 of phenol. Potential scan rate 10 mV s1. (C) Scheme of in situ HRPMesoporous material in which all the enzyme molecules are isolated in ideal meso-cages. (D) Scheme of the scenario in HRP/SBA-15 electrode, in which two types of HRP molecules are presented: some inside the channels and some outside the channels.
E. Santalla et al. / Solid State Sciences 13 (2011) 691e697
The results obtained show that the immobilization process in the case of HRP post-grafted on SBA-15 is efficient, but not very selective in terms of confinement. On other hand the in-situ immobilization of HRP in mesoporous materials yields enzyme molecules that are successfully isolated by the silica, i.e., encapsulated in isolated cages in meoporous material as represented in the scheme (Fig. 6B). Therefore, we can conclude that the encapsulation method using surfactant to assist the synthesis of a mesoporous material has worked successfully since enzyme keeps “alive” and in this case also it stays confined selectively inside mesoporous cages. 4. Conclusions From the results observed and discussed we can conclude that the parameters influencing the in situ synthesis of ordered mesoporous biocatalysts are the following: Hydrophobicity of the surface of the protein: If the protein is hydrophobic enough the siliceous material is synthesized even in the presence of electrostatic charges. When the protein is charged, the relevance of this depends on the protein surface. If the surface is hydrophobic enough, the hydrophobic interactions prevail and the right structure is formed. If the surface is hydrophilic then the charges may be the driving force of the process, and no order is obtained in the final material. The surfactant cannot form the micelles properly and even nonporous nanostructures can be formed. The relevance of these results relies on the fact that they help us to understand the complex process and to envisage parameters that
697
should be studied in order to reach a methodology enabling to establish a general protocol to get the synthesis of OMM using any enzyme.
Acknowledgements The authors acknowledge the Spanish Ministry of Education and Science for the financial support (MAT-2006-04107).
References [1] J.L. Blin, C. Gérardin, C. Carteret, L. Rodehüser, C. Selve, M.J. Stébé, Chem. Mater. 17 (2005) 1479. [2] M. Mureseanu, A. Galarneau, G. Renard, F. Fajula, Langmu 21 (2005) 4648. [3] Y. Wei, H. Dong, J. Xu, Q. Feng Chem, Phys. Chem. 9 (2002) 802. [4] X.Y. Yang, Z.Q. Li, B. Liu, A. Klein-Hofmann, G. Tian, Y.F. Feng, et al., Adv. Mater. 18 (2006) 410. [5] E. Serra, V. Alfredsson, R.M. Blanco, I. Díaz, Stud. Surf. Sci. Catal. 174 (2008) 369. [6] E. Serra, A. Mayoral, Y. Sakamoto, R.M. Blanco, I. Díaz, Micropor. Mesopor. Mater. 114 (2008) 201e213. [7] E. Serra, R.M. Blanco, V. Alfredsson, I. Díaz, Micropor. Mesopor. Mater. 129 (2010) 173e178. [8] Y. Wan, D.Y. Zhao, Chem. Rev. 107 (2007) 2821. [9] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [10] B.J. Davis, N.Y. Ann, Acad. Sci. 121 (1964) 404. [11] Y. Sakamoto, I. Díaz, O. Terasaki, D. Zhao, J. Pérez-Pariente, J.M. Kim, et al., Chem. B. 106 (2002) 3118. [12] I. Díaz, B. García, B. Alonso, C.M. Casado, M. Morán, J. Losada, et al., Chem. Mater. 15 (2003) 1073. [13] S.S. Rosatto, L.T. Kubota, G. de Oliveira Neto, Anal. Chim. Acta 390 (1999) 65.