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J. Phys. Chem. C 2007, 111, 3842-3847
Novel Synthesis of Ferric Impregnated Silica Nanoparticles and Their Evaluation as a Matrix for Enzyme Immobilization R. S. Prakasham,*,† G. Sarala Devi,*,‡ K. Rajya Laxmi,† and Ch. Subba Rao† Bioengineering and EnVironmental Centre and Inorganic and Physical Chemistry DiVision, Indian Institute of Chemical Technology, Taranaka, Hyderabad 500 007, India ReceiVed: October 26, 2006; In Final Form: December 6, 2006
Silica nanoparticles were synthesized by sol-gel route using a cationic surfactant, tetrabutylammonium bromide (TBAB), as a templating agent and a nonionic surfactant block copolymer [poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)] to regulate the silicon oxide grain size. Ferric oxide (Fe2O3) was impregnated to these silica nanoparticles by wet impregnation method. The diameter of the ferric impregnated silicon oxide particle (FSP) was within 10 nm. The nature of ferric species (Fe+3) in FSP was confirmed by X-ray diffraction (XRD) and FT-IR studies. Diastase enzyme was immobilized onto FSP, and its presence was evidenced by the appearance of the N1s peak in the XPS spectra. Scanning electron microscopic and transmission electron microscopic studies revealed the presence of enzyme on the surface of FSP. The enzyme immobilized particles (EI-FSP) were characterized for biocatalytic activity and kinetic behavior of bound diastase enzyme. The physiological environment (the temperature and pH of the reaction mixture) influenced the nature of the enzyme binding. Reusability studies indicated that this EI-FSP could be used more than 50 cycles without any significant loss of enzyme activity. This is the first report of its kind in preparation of ferric impregnated silicon oxide nanoparticles and its application as matrix material for enzyme immobilization for biocatalyst reusability.
1. Introduction Mesoporous materials have opened many new possibilities for application in catalysis,1 chromatographic separation,2 and nanoscience due to their tunable pore size and large surface area.3-5 It is apparent that morphology control as well as handling the texture of mesoporous materials is extremely important for many industrial applications.6-10 Hence, more recently, the mesoporous silica with different morphologies such as thin films, monoliths, hexagonal prisms, and hollow tubular spheres have been synthesized.11 Moreover, nanoparticle pore size can be tunable during synthesis by changing the molecular structure of the surfactant. In general, mesoporous silica as such is a poor immobilization matrix especially for biocatalysts. Preparation of biocompatible surfaces for immobilization of enzymes and whole cells12 is an important aspect of biotechnology. Hence, synthesis of ionic nature mesoporous silica has been reported by using mixed cationic-nonionic surfactant in rare cases.13 The immobilization of enzymes onto insoluble supports has been a topic of active research in enzyme technology and is an important tool for their application at industrial processes, fabrication of a diverse range of functional materials or devices.14 Immobilization provides many advantages, such as use in continuous operations, product purification, catalyst recycling, enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth.15,16 Several immobiliza* Corresponding author. Fax: +91-40-27193159 (R.S.P.); +91-4027160921 (G.S.D.). E-mail:
[email protected] (R.S.P.);
[email protected],
[email protected] (G.S.D.). † Bioengineering and Environmental Centre. ‡ Inorganic and Physical Chemistry Division.
tion techniques have been developed on the basis of the interaction between the biomolecule to be immobilized and support material and evaluated in detail.17 However, these immobilization techniques use either natural or synthetic polymers or chemical as matrices.17 The recent development in this sector is use of nanosize materials as supports for whole cell or enzyme immobilization.13,18 Among these materials magnetic nanoparticles became very popular when used in conjunction with biological materials such as proteins, peptides, enzymes, antibodies, and nucleic acids because of their unique properties.19-23 The magnetic ion impregnated silica base provides a good degree of biocompatibility and a high specific area whose rich chemistry allows easy functionalization and immobilization of biomolecules.13,24 Herein we present a novel sol-gel confinement route for the design and synthesis of ferric impregnated silica particles (FSP) and characterized the surface chemistry. These particles were evaluated as matrix material by immobilizing the diastase enzyme and evaluating its binding, kinetic properties, and reusability. 2. Experimental Methods 2.1. Synthesis of Ferric Impregnated Silicon Oxide Particles. Silica nanoparticles were synthesized via sol-gel route under basic condition by the modified method of Suzuki et al.25 In a typical procedure, 2.6 g of tetrabutylammonium bromide (TBAB) and 1.33 g of block copolymer [poly(ethylene glycol)block-poly(propylene glycol)-block-poly(ethylene glycol)] were dissolved in 150 mL of dilute HCl solution under stirring at room temperature. To this solution, 3.7 mL of tetraethylorthosilicate (TEOS) was added dropwise and incubated under constant shaking environment. After 24 h, the sol was neutralized using dilute NH3 solution. This gel was aged at room
10.1021/jp0670182 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/15/2007
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Figure 2. Influence of medium pH on enzyme loading onto FSP.
Figure 3. XRD pattern of FSP and EI-FSP.
Figure 1. Schematic representation of sol-gel synthesis of ferric coated silicon oxide nanoparticles.
temperature for 24 h and oven-dried at 70 °C. The resultant material was calcined at 600 °C to get silica nanoparticles. These ordered nanoparticles were impregnated with ferric ions using FeCl3 in a silica/iron molar ratio of 1:4 and then calcined at 300 °C to get iron oxide coated silica nanoparticles, as shown in Figure 1 and used for characterization and enzyme immobilization studies. The acidic condition promotes hydration of TEOS and decreases the residual ethoxy group affecting the assembly of the particles; subsequent addition of base (ammonia solution) was effective in the formation of ordered nanoparticles through self-assembly of negatively charged silicate and positively charged (TBAB) miceller and nonionic co-block polymer because the polarity decreased with the assembly of the anionic and cationic species, and co-block polymer nonionic surfactant surrounds the silica-TBAB composite due to weak interaction between ionic and nonionic hydrophilic groups. Thus, the nonionic surfactant acts as a grain growth inhibitor and stabilizes the ordered nanoparticles. 2.2. Preparation of Enzyme Impregnated FSP. Commercially available diastase enzyme (Sigma-Aldrich, USA) was used in this investigation. A 100 mg amount of enzyme was dissolved in 1 mL of distilled water, and to this 100 mg of
synthesized matrix particles was added under nitrogen atmosphere. This mixture was incubated at 30 °C for 30 min under constant mixing environment. The contents were then centrifuged at 10 000 rpm for 5 min at 30 °C. The unbound enzyme from these particles was removed by washing with sterilized distilled water until the supernatant was free of protein and/or enzyme activity. These enzyme impregnated FSP (EI-FSP) were dried using a vacuum evaporator and used for characterization and evaluation of its biological activity. 2.3. Characterization of FSP and EI-FSP. FSP and EIFSP were characterized for their surface chemistry and morphology to understand the nature of physicochemical properties and enzyme binding using different analytical techniques (XRD, XPS, and FT-IR, etc.). X-ray Diffraction (XRD) measurement before and after enzyme bound FSP mounted onto quartz substrate was carried out on D/8 BRUKER AXS operating at 40 kV at a current of 30 mA with Cu KR radiation. FT-IR studies were conducted using a Thermo Nicolet Nexus 670 spectrometer. Demoisturized samples before and after enzyme binding (1-2 mg) were homogenized with 100 mg of dry KBr and made into pellets. These pellets were analyzed for transmittance in the range of 4000 to 400 cm-1. Surface area and pore size distribution were measured by a Brauner-Emmet-Teller (BET) method using N2 sorption isotherm. X-ray photoelectron spectroscopy (XPS) measurements were obtained on a KRATOS-AXIS 165 instrument equipped with dual aluminum-magnesium anodes using Mg KR radiation (the X-ray power supplied was 15 kV and 5
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Figure 5. Pore size distribution of FSP particles.
3. Results and Discussion
Figure 4. FT-IR spectra of FSP and EI-FSP.
mA at 10-9 Torr pressure). The peak positions were based on calibration with respect to the “C 1s” peak at 284.6 eV. The obtained XPS spectra were fitted using a nonlinear square method with the convolution of Lorentzian and Gaussian functions after the polynomial background subtraction from the raw data. For microscopic studies, different materials (FSP and EI-FSP) were fixed by incubating in 2% aqueous osmium tetraoxide for 2 h. The samples were then dehydrated using graded alcohol in series and dried to critical point by incubating in an Electron Microscopy Science CPD unit. Scanning electron microscopy (SEM; model, HITACHI) and transmission electron microscopy (TEM; TECH NAI-12, operated at 120 KeV) were used to carry out the morphological studies. 2.4. Enzyme Activity Measurement. The enzyme activity was measured according to ref 26 by estimating the maltose produced during starch hydrolysis using 3,5-dinitrosalicylic acid as a coupling reagent. The reaction mixture containing 1.0 mL of 1% soluble starch in 0.01 M phosphate buffer (pH 5.0) and 10 mg of EI-FSP was incubated at 30 °C for 5 min and centrifuged to separate the EI-FSP and the reaction mixture. The reaction was stopped by adding 2.0 mL of 3,5-dinitrosalicylic acid solution followed by heating in a boiling water bath for 5 min. The contents were then cooled to room temperature, and the volume was made up to 10 mL with distilled water. The absorbance of the reaction mixture was determined at 540 nm in a UV-visible spectrophotometer. The temperature effect on the enzyme was studied by performing the reaction at predetermined temperature. Reusability studies were performed by washing the EI-FSP particles after every reaction with distilled water. One unit of the enzyme was defined as the amount of enzyme capable of producing 1 M reducing sugar (as maltose) from 1% soluble starch as substrate in 1 min at 30 °C. 2.5. Calculation of Km, Vmax, Activation Energy Values, and Protein Estimation. Reaction kinetics for EI-FSP and free enzyme were estimated by measuring the catalytic activity at different substrate concentrations ranging from 0.5 to 3.0% at 40 °C. Km and Vmax values were calculated by representing the enzyme activity values at different substrate concentrations in a Lineweaver-Burk equation. The protein content of the enzyme was estimated according to the method of Lowry et al.27
3.1. Enzyme Binding Studies. The diastase enzyme-binding pattern with respect to solution pH was investigated by using a 1:1 ratio of FSP and enzyme (w/w). After thorough mixing for 15 min at room temperature, the contents were centrifuged to remove the unbound enzyme present in the solution. The enzyme concentration in the supernatant was determined in terms of protein content, and the immobilized enzyme was calculated by deducting the enzyme present in the supernatant from the enzyme solution before binding studies. The effect of pH on the enzyme loading was investigated in the range of pH 3-7, as shown in Figure 2. It was observed that enzyme loading was higher in the range of pH 3-5 (the maximum loading (41%) at pH 5.0) and further increase caused drastic reduction (6-8 times) in enzyme loading. Figure 3 gives the wide-angle XRD pattern of FSP and EIFSP, where both samples revealed a typical XRD pattern showing strong diffraction peaks 2θ at 33, 35.5, 49.2, 54.2, 62, and 63.5° corresponding to the 104, 110, 024, 116, 214, and 300 planes, respectively, matching well with the standard data for R-Fe2O3 indicating the hematite presence in the samples (Figure 3). No peaks were noticed for SiO2 presence in either samples, suggesting silica is not present on the surface of the particles; rather it might be covered by Fe2O3 or it may not be in crystallized form, as observed similar to the studies done by Tago et al.24 The results in the present investigation coincide with the observations made by Lo and Chen,28 where the authors observed hematite at 150 °C and above 150 °C. The decrease in peak intensities (∼50%) after enzyme binding may be due to the surface binding of the enzyme, suggesting the indirect evidence for enzyme incorporation in these particles (Figure 3). Figure 4 gives the FT-IR spectra recorded for FSP and EIFSP nanoparticles. FT-IR analysis showed two prominent peaks, one at 470 cm-1 and another at 546 cm-1 in both samples. These two absorption bands can be correlated to characteristic excitation of Fe2O3 and SiO2 vibrations, suggesting the presence of these two elements in these nanopartices. However, a slight shift in these two peaks was noticed with the EI-FSP samples (Figure 4). In addition, the peaks at 803, 1089, and 1627 cm-1 also correspond to SiO2 vibration. The resence of the peak at 2924 cm-1 in the EI-FSP samples does suggest methylene antisymmetic and symmetric vibration, which may be due to the enzyme presence. This was further evidenced from an intense OH stretching band in the range of 3420-3436 cm-1 after enzyme binding (Figure 4), depicting the presence of enzyme on the surface of the particles. The surface area, particle size, and pore size distribution were analyzed by BET method using nitrogen adsorption-desorption
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Figure 6. XPS spectra of FSP and EI-FSP for various oxidation states of elements present on the surface of the particles.
Figure 7. Scanning electron micrographs of FSP and EI-FSP.
Figure 8. Transmission electron micrographs of FSP and EI-FSP.
isotherms. The FSP showed larger pore ranging from 0.6 nm attributed to interparticle space among nanoparticles and sharp distribution around 4 nm due to ordered arrangement (Figure 5). Furthermore, the data depicted that the surface area and pore volume of the EI-FSP decreased from 47.4 to 44.0 m2 g-1 and from 0.0048 to 0.0037 Å3/g, respectively, suggesting the enzyme
immobilization within the channels of the nanoscale FSP. These data were consistent with the literature.29 The large pore volume along with the smaller particle size of FSP makes it easy for the diffusion and adsorption of biomolecules (enzyme). XPS depicts the stoichiometry of constituent elements (oxygen, iron, silica, carbon, and nitrogen) present in FSP and
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Prakasham et al. resulted in drastic reduction of biocatalytic activity (>64%) (results not shown). The maximum activity (8040 units/(µg of protein)) was observed at 40 °C. Therefore, immobilized enzyme kinetics was investigated at 40 °C. The Km, Vmax, and activation energy values calculated for EI-FSP were observed to be 1.818 mM, 40 000 U/(µg of protein), and 16 325.51 kcal/mol, respectively. Reusability studies were performed at 40 °C and at pH 5.0 environment. The data revealed that the immobilized enzyme was stable and showed starch degradation activity for more than 50 cycles with a variation 5% activity (results not shown). 4. Conclusions
Figure 9. Effect of reaction medium pH on immobilized enzyme activity.
EI-FSP and information about the chemical environment (Figure 6). The peaks at 532 and 534 eV attributed to O2- and O22species, respectively, are in close agreement with the literature value.30 The Fe 2p spectrum revealed spin-orbit splitting of Fe 2p3/2 ground state at 711.9 eV for FSP and 712 eV for EIFSP, whereas the Fe 2p1/2 excited state was observed at 726 eV for FSP and 725.6 eV for EI-FSP. These peaks were attributed to Fe+3 in Fe2O3, respectively. The peak at 102 eV corresponds to the Si 2p level. Typical deconvolution of C 1s spectra (with characteristic peaks at 284.5, 286.6, and 288.4 eV attributed to -C-C-, -C-O-, and -CdO-, respectively) present only in EI-FSP further confirmed the presence of enzyme. Apart from these, the N 1s peak at 400 eV only in the EI-FSP sample due to the presence of the NH2 group on the surface further provides the evidence for enzyme binding on the surface. SEM studies were performed for FSP and EI-FSP to determine the morphology and size distribution. EI-FSP were thoroughly washed to remove the unbound enzyme, dried at room temperature under vacuum, and subjected to SEM studies. A patterned structure of paramagnetic particles before and after enzyme loading was noticed. Immobilized enzyme presence on these paramagnetic particles also could be seen (Figure 7). Figure 8 gives the TEM photographs of FSP and EI-FSP. It was observed that there was little change in particle size before and after enzyme immobilization and the average particle size is ∼10 nm. The size of the EI-FSP was further confirmed by BET analysis. These studies further reveal that the interaction of enzyme with FSP particles was through the hydrophobic nature. This was also confirmed on the basis of the fact that the enzyme was not washed out under hydrophilic environment as the unbound enzyme was removed by repeated washing using distilled water. 3.2. Biocatalytic Evaluation of EI-FSP. The diastase enzyme impregnated on FSP was evaluated for starch hydrolysis function at different temperature conditions and at different pH environments. It was noticed that the medium pH and temperature influenced the biocatalytic activity of the immobilized enzyme. Maxium enzyme activity (6800 units/(µg of protein)) was noticed at pH 5.0 environment and any variation in medium pH drastically reduced the biocatalytic activity (Figure 9). The enzyme activity inhibition was more than 60% with the change of one pH unit toward neutrality. However, decrease of medium pH from 5.0 to 4.0 resulted in inhibition of biocatalytic activity more than 80% (Figure 9). Immobilized enzyme activity varied with reaction medium temperature. Only slight variation in enzyme activity was observed with the variation of reaction mixture temperature from 30 to 40 °C and further increase
We successfully synthesized ferric-silica nanoparticles (FSP) of approximately 10 nm using binary surfactant, which performed a function as template and as a grain growth inhibitor. XRD studies confirm that the synthesized FSP contain Fe2O3. These nanoparticles were used as a matrix for enzyme diastase immobilization. Confirmation of the enzyme binding was demonstrated by FTIR and XPS studies; the sizes of the particles were characterized by BET and TEM studies. Enzyme binding at different pH environments revealed that enzyme loading differs with the pH of the reaction medium. Higher enzyme loading was observed in the range of pH 3-5, and further increase in the reaction solution pH (6-7) caused drastic reduction (6-8 times) in enzyme loading. Reusability studies revealed the EI-FSP particles could be used for more than 50 cycles without loss of enzyme activity. Kinetic data indicated that the catalysis occurs at lower activation energy levels with immobilized enzyme compared to free enzyme, indicating the economic importance of this EI-FSP in reusability and economizing the process. Acknowledgment. The authors thank the Department of Science and Technology, New Delhi, for financial support. Supporting Information Available: Information regarding chemicals procured and used for synthesis of FSP. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Tanev, P. T.; Chibwe, M.; Pinnavaia, T. J. Nature 1994, 368, 321. (2) Liewellyn, P. L.; Ciesla, U.; Decher, H. Stud. Surf. Sci. Catal. 1994, 84, 2013. (3) Wu, C. G.; Bein, T. Chem. Mater. 1994, 6, 1109. (4) Agger, J. R.; Anderson, M. W.; Pemble, M. E. J. Phys. Chem. B 1998, 102, 3345. (5) Corma, A. Chem. ReV. 1997, 97, 237. (6) Lee, T.; Yao, N.; Aksay, I. A. Langmuir 1997, 13, 3866. (7) Zhao, D.; Huo, Q.; Feng, J.; Chemelka, B. F.; Stuck, G. D. J. Am. Chem. Soc. 1998, 120, 6024. (8) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, N.; Chmelka, B. F.; Stucky, G. D. Science 1998, 382, 548. (9) Mann, S.; Ozin, G. A. Nature 1996, 382, 313. (10) Yang, S. M.; Yang, H.; Coombs, N.; Sokolov, I.; Kresge, C. T.; Ozin, G. A. AdV. Mater. 1999, 11, 52. (11) Yong, H.; Coombs, N.; Ozin, G. A. Nature 1997, 386, 692. (12) Kouassi, G. K.; Irudayaraj, J.; Gregory, Mc Carty. Biomagn. Res. Technol. 2005, 3 (1), 1. (13) Ryoo, R.; Joo, S. H.; Kim, J. M. J. Phys. Chem. B 1999, 103, 7435. (14) Tischer, W.; Wedekind, F. Top Curr. Chem. 1999, 200, 95. (15) Jia, H.; Guangyu, Z.; Wang, P. Biotechnol. Bioeng. 2003, 84, 407. (16) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336. (17) Ramakrishna, S. V.; Prakasham, R. S. Curr. Sci. 1999, 77, 87. (18) Phadtare, S.; Parekh, P.; Tambe, S. S. A.; Joshi, R.; Sainkar, S. R.; Prabhhune, A.; Sastry, M. Biotechnol. Prog. 2003, 19, 1659. (19) Liao, M. H.; Chen, D. H. Biotechnol. Lett. 2001, 23, 1723. (20) Huang, S. H.; Liao, M. H.; Chen, D. H. Biotechnol. Prog. 2003, 19, 1095.
Ferric Impregnated Silica Nanoparticles (21) Koneracka, M.; Kopcansky, P.; Antalik, M.; Timko, M.; Ramchand, C. N.; Lobo, D.; Mehta, R.; Upadhyay, R. V. J. Magn. Mater. 1999, 201, 427. (22) Konda, A.; Fukuda, H. J. Ferment. Bioeng. 1997, 84, 337. (23) Wilheim, C.; Gazeau, F.; Roger, J.; Pons, N.; Salis, M. F.; Perzynski, R. Phys. ReV. E 2002, 65, 31404. (24) Tago, T.; Hatmta, T.; Miyajima, K.; Kishida, M.; Tashiro, S.; Wakbayashi, K. J. Am. Ceram. Soc. 2002, 85, 2188. (25) Suzuki, K.; Ikari, K.; Imai, H. J. Am. Chem. Soc. 2004, 126, 462.
J. Phys. Chem. C, Vol. 111, No. 10, 2007 3847 (26) Bernfeld, P. Enzymes of Starch Degradation and Synthesis. In AdVances in Enzymology; Nord, F. F., Ed.; Interscience: New York, 1951; Vol. 12, pp 385-386. (27) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (28) Lo, S. L.; Chen, T. Y. Chemosphere 1997, 35, 919. (29) Sun, J.; Zhang, H.; Tian, R.; Ma, D.; Bao, X.; Su, D. S.; Zou, H. Chem. Commun. (Cambridge, U.K.) 2006, 1322. (30) Handbook of X-ray Photoelectron Spectroscopy; Wagner, C. D., Ed.; Perkin Elmer: Eden Prairie, MN, 1979.
