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Physical and Chemical Adsorption of Mucor javanicus Lipase on SBA-15 Mesoporous Silica. Synthesis, Structural Characterization, and Activity Performance Andrea Salis, Daniela Meloni, Stefania Ligas, Maria F. Casula, Maura Monduzzi,* Vincenzo Solinas, and Emil Dumitriu† Department of Chemical Sciences, University of Cagliari, S.S. 554 Bivio Sestu 09042 Monserrato (CA), Italy Received November 11, 2004. In Final Form: February 28, 2005 In this work a sample of SBA-15 mesoporous silica was synthesized and characterized by TEM, XRD, and N2 adsorption. The sample had a high value of specific surface area (1007 m2 g-1) and total pore volume (2.1 cm3 g-1). The pore diameter was 67 Å, so it was large enough to accommodate protein molecules inside the channels. Immobilization by physical adsorption of a commercial lipase preparation from Mucor javanicus was performed at different pH values (pH 5-8). pH 6 gave the highest lipase loading and hydrolytic activity of the corresponding biocatalyst. Chemical modification of the SBA-15 via glutardialdehyde allowed also the enzyme immobilization through chemical adsorption. This preparation was active toward tributyrin hydrolysis. On the contrary, very low activity toward triolein hydrolysis was observed. The reduction of the size of the channels due the immobilization process has been suggested as a possible explanation.
1. Introduction Silica-based porous materials, because of their high surface area and tunable pore diameter, are regarded as suitable hosts for large molecules such as proteins.1 Protein entrapment in sol-gel matrixes for potential applications in the field of biosensors has been extensively studied.2,3 The lack of a precise mesopore size control4 in such solgel materials, however, limits the possibility of selectively isolating specific proteins. On the other hand, ordered mesoporous silica with a well-defined pore size such as MCM-41 is not suitable, since bulkier enzymes (diameter > 40 Å) cannot access the pores. Mesoporous silica with uniform large channels and high stability, namely SBA-15, has recently been synthesized.5,6 The synthesis makes use of triblock copolymers as structure-directing agents for silica.7 This allows for the accurate control of the porous structure over a wide range of pore size (50-300 Å). These materials can be synthesized by tuning their selectivity toward specific features of proteins, such as dimensions and electric charge, by varying the pore diameter and by derivatizing the reactive silanol groups with suitable functionalized organic silanes. Applications of these materials range from selective sequestration to release of proteins.8 When the hosted protein in the mesoporous material is an enzyme, an immobilized biocatalyst is obtained. The * Corresponding author. Tel: +39-070-675-4385. Fax: +39-070675-4388. E-mail:
[email protected]. † Present address: Laboratory of Catalysis, Technical University of Iasi, 71 D. 700050, Mangeron, Iasi, Romania. (1) Moller, K.; Bein, T. Chem. Mater. 1998, 10, 2950. (2) Wang, J. Anal. Chim. Acta 1999, 399, 21. (3) Dong, S.; Chen, X. Rev. Mol. Biotechnol. 2002, 82, 303. (4) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120. (5) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G. H.; Chmelka, B. F.; Stucky, G. D. Science 1998, 279, 548. (6) Konya, Z.; Zhu, J.; Szegedi, A.; Kiricsi, I.; Alivisatos, P.; Somorjai, G. A. Chem. Commun. 2003, 3, 314. (7) Kipkemboi, P.; Fogden, A.; Alfredsson, V.; Foldstrom, K. Langmuir 2001, 17, 5398. (8) Han, Y.-J.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1999, 121, 9897.
preparation of an immobilized biocatalyst is of great interest due to some important advantages, such as: (1) increase of the operational stability of the enzyme; (2) easy recovery (for instance, by simple filtration) of the immobilized enzyme at the end of the reaction, and thus its potential reuse; and (3) accomplishment of the enzyme-catalyzed reaction under a continuous operation. The immobilization on SBA-15 of different protein and enzymes, such as conalbumin,9 cytochrome C,10 horseradish peroxidase,11,12 subtilisin,12 trypsin,13,14 R-amylase,15 chloroperoxidase,16 and lysozyme,17 has been recently investigated. Among various enzymes, lipases are of great interest owing to their properties and applications. Lipases are ubiquitous enzymes that in nature catalyze triacylglycerol hydrolysis. They are the most used enzymes for biotechnological applications both for large-scale processes and for the fine chemical syntheses.18 Moreover, lipases show high chemo-, regio- and stereoselectivity and can be used in organic media. They have become powerful tools for the organic chemists. Very recent works investigated lipase immobilization on M41S19 and MCM-3620 meso(9) Han, Y.-J.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1999, 121, 9897. (10) Washmon-Kriel, L.; Jimenez, V. L.; K. J. B., Jr. J. Mol. Catal. B 2000, 10, 453. (11) Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S. Chem. Mater. 2000, 12, 3301. (12) Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S. Microporous Mesoporous Mater. 2001, 44-45, 755. (13) Yiu, H. P.; Wright, P. A.; Botting, N. P. Microporous Mesoporous Mater. 2001, 44-45, 763. (14) Yiu, H. P.; Wright, P. A.; Botting, N. P. J. Mol. Catal. B 2001, 15, 81. (15) Mody, H. M.; Mody, K. H.; Jasra, R. V. Indian J. Chem. 2002, 41, 1795. (16) Han, Y.-J.; Watson, J. T.; Stucky, G. D.; Butler, A. J. Mol. Catal. B 2002, 17, 1. (17) Vinu, A.; Murugesan, V.; Hartmann, M. J. Phys. Chem. B 2004, 108, 7323. (18) Jaeger, K. E.; Eggert, T. Curr. Opin. Biotechnol. 2002, 13, 390. (19) Macario, A.; Calabro`, V.; Curcio, S.; Paola, M. D.; Giordano, G.; Iorio, G.; Katovic, A. Stud. Surf. Sci. Catal. 2002, 142, 1561. (20) Dumitriu, E.; Secundo, F.; Patarin, J.; Fechete, I. J. Mol. Catal. B 2003, 22, 119.
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porous materials and on SBA-15 mesoporous silica in two cases.21,22 Particularly, the physical immobilization on SBA-15 by adsorption of Newlase F, which contains lipase and acid protease from Rubus niveus,21 was used for the enzymatic hydrolysis of protected dipeptide alkyl esters (n-heptyl). In addition, SBA-15 and other materials-such as metal oxides and ceramics-were used to immobilize lipase PS (Pseudomonas cepacia) as biocatalysts for the enantioselective acetylation of methyl (()-mandelate in an ionic liquid solvent system.22 The present work deals with the synthesis and characterization of SBA-15 mesoporous silica and its use as a support for the immobilization of a commercial lipase obtained from Mucor javanicus. The effect of the pH of the immobilization solution on lipase loading as well as on the catalytic activity of the corresponding preparation was studied. Immobilization by chemical adsorption via glutardialdehyde was also carried out and the activity of this preparation was compared to the free and the physically adsorbed lipase. 2. Materials and Methods 2.1. Chemicals. Lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3) from M. javanicus was purchased from Amano Enzymes. This lipase is a commercial preparation with a protein content of about 20% (w/w). Tributyrin 98%, triolein 65%, sodium deoxycholate, and gum arabic were from Sigma (St. Louis, MO). Pluronic copolymer 123 (EO20PO70EO20) was purchased from Aldrich. 2.2. Synthesis of SBA-15. SBA-15 samples were prepared by using the templating effect of nonionic triblock copolymer surfactants according to a previous report.5 Briefly, 4 g of Pluronic copolymer 123 was dissolved in 20 mL of 37 wt % HCl and 120 mL of distilled water; the resulting mixture was stirred at 308 K for 16 h. Then, 8.5 g of tetraethyl orthosilicate (TEOS) was added and the final solution was stirred at 308 K for 24 h. Finally, the mixture was crystallized into a Teflon-lined autoclave at 373 K for 24 h. After filtration and washing, the solid was dried at 313 K and then calcined at 823 K for 5 h. 2.3. Characterization of SBA-15. Transmission electron microscopy (TEM) images were obtained on a JEOL 200CX microscope equipped with a tungsten cathode operating at 200 kV. Finely ground SBA-15 samples were dispersed in n-octane by sonication and dropped and dried on a carbon-coated copper grid for observations. X-ray diffraction (XRD) spectra were recorded on a X3000 Seifert diffractometer equipped with Cu KR radiation and with a graphite monochromator on the diffracted beam. Textural analysis of the SBA-15 silica matrix was carried out on a Thermoquest-Sorptomatic 1990, by determining the N2 adsorption/desorption isotherms at 77 K. Before analysis, the samples were heated to 523 K at a rate of 1 K min-1 under vacuum. The specific surface area, the total pore volume, and the pore size distribution were assessed by the Brunauer-Emmett-Teller (BET)23 and the Dollimore-Heal (DH) method, respectively.24 2.4. Lipase Activity Assays. Tributyrin and triolein hydrolysis were measured by the pH-stat method,25,26 using a 718 Stat Titrino equipment from Metrohm (Herisau, Switzerland). Tributyrin Assay. A sample of 250 µL of lipase in water solution (or 20-40 mg of the immobilized preparation) was added to a gum arabic-stabilized emulsion of tributyrin in distilled water at 25 °C. The pH was maintained at 7.0 by titration with 10 mM sodium hydroxide solution. The substrate emulsion was prepared by homogenizing a mixture of tributyrin (3 mL), distilled water (21) Chen, Z.-Z.; Li, Y.-M.; Peng, X.; Huang, F.-R.; Zhao, Y.-F. J. Mol. Catal. B 2002, 18, 243. (22) Itoh, T.; Ouchi, N.; Nishimura, Y.; Hui, H. S.; Katada, N.; Niwa, M.; Onaka, M. Green Chem. 2003, 5, 494. (23) Brumaner, S.; Emmet, P. H.; Teller, E. J. Am. Chem. Soc. 1938, 60, 309. (24) Dollimore, D.; Heal, G. R. J. Appl. Chem. 1964, 14, 109. (25) Bosley, J. A.; Peilow, A. D. J. Am. Oil Chem. Soc. 1997, 74, 107. (26) Bloomer, S.; Adlercreutz, P.; Mattiasson, B. J. Am. Oil Chem. Soc. 1990, 67, 519.
Salis et al. (47 mL), and an emulsification reagent (10 mL) at 18 000 rpm for 1 min by an Ultra-Turrax homogenizer. The emulsification reagent was prepared by dissolving gum arabic (6.0 g), glycerol (54 mL), NaCl (1.79 g), and KH2PO4 (0.041 g) in distilled water (40 mL). Triolein Assay. The assay mixture contained 5 mL of the substrate emulsion, 50 µL of 10 mM sodium deoxycholate, and 0.89% NaCl solution added to reach the final volume of 10 mL. The pH was adjusted to 7 by the addition of 10 mM NaOH, and 250 µL of lipase solution (40 mg lipase dissolved in 0.89% NaCl) or 20-40 mg of the immobilized preparation was added. The continuously stirred mixture was incubated at 25 °C. The release of oleic acid was monitored by titration with 10 mM NaOH in the pH-stat equipment. The substrate emulsion was prepared by mixing 500 mg of triolein 65%, 9.5 mL of 0.89% NaCl solution, and 500 mg of gum arabic (added slowly to the liquids during Ultra Turrax mixing). Mixing was continued for 1 min and the emulsion was used immediately. 2.5. Enzyme Immobilization by Physical Adsorption. Enzyme support, SBA-15 (100 mg), was placed in 10 mL capped vials. The vials were filled with 8 mL of a filtered enzymatic solution 5 mg mL-1 in 20 mM sodium phosphate buffer (pH 5, 6, 7, and 8). All the vials were slowly rotated for 5 h at 298 K. The suspension was centrifuged and the mother liquor was removed from each vial. The solid was washed with 1 mL of buffer and centrifuged and the liquid removed. This procedure was repeated twice. The mother liquor and the resulted washing solutions were collected together and checked for residual activity with the tributyrin assay. The immobilized lipases were dried overnight (at room temperature) under vacuum. To follow the dynamic evolution of the physical adsorption process, samples of 100 µL were withdrawn, at established times, from the vessel containing the enzymatic solution and the SBA15 solid powder. The samples were analyzed by the tributyrin assay. 2.6. Chemical Modification of SBA-15. The functionalization of mesoporous silica SBA-15 was carried out as follows. Mesoporous silica (4 g) was treated with 2% v/v 3-aminopropyltriethoxysilane solution in dry toluene (40 mL). The mixture was heated under reflux and under an inert nitrogen atmosphere for 15 h. The aminosilylated material was collected by filtration, washed with acetone, and dried overnight at 80 °C under vacuum. Glutardialdehyde-activated SBA-15 was prepared by soaking aminosilylated mesoporous silica (2.5 g) in a mixture of 2 mL of 25% aqueous glutardialdehyde and 18 mL of 0.1 M phosphate buffer solution (pH 7.5) for 1 h. Then, the chemically modified SBA-15 was washed with 100 mL of water and finally with 100 mL of the same buffer on a sintered glass filter. 2.7. Enzyme Immobilization by Chemical Adsorption. About 1 g of crude lipase from M. javanicus was slowly dissolved in 20 mL of 0.1 M pH 8 phosphate buffer in an ice bath. The resulting solution was filtered to eliminate undissolved particles and diluted to the final volume of 25 mL. A volume of 8 mL of the lipase solution was added to the chemically modified SBA15, and the resulting suspension was slowly rotated overnight. The suspension was filtered, washed with the buffer and 2-propanol, and dried under vacuum. 2.8. Molecular Modeling. The geometries of tributyrin and triolein molecules were optimized in vacuo using the MMFF94 force field as implemented in Spartan version 5.1.3.27
3. Results and Discussion 3.1. Structure of the SBA-15 Mesoporous Silica. The morphological and structural characterization of the SBA-15 mesoporous silica was performed by the combined use of TEM, XRD, and N2 physisorption techniques. Typical TEM images of the SBA-15 sample are shown in parts a and b of Figure 1, which represent a side view and a top view of the sample, respectively. Figure 1a clearly shows the presence of ordered arrays of silica channels with uniform diameter (around 6 nm) and wall thickness (around 3 nm). As indicated by the top view micrograph, (27) SPARTAN, W., Inc. Version 5.1.3; 18401 Von Karman, Suite 370, Irvine, CA.
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hysteresis at high relative pressure.28 These are typical features of hexagonal cylindrical channel mesoporous materials. In particular, it is noteworthy that an H1 hysteresis indicates the presence of open channels. This is important for catalytic applications, which require an effective diffusion of the reactants through the porous materials. Figure 2b shows the very narrow pore size distribution (determined from desorption branch) around the pore diameter mean value of 67 Å, in agreement with TEM observations. A very large surface area of 1007 m2 g-1 and a total pore volume of 2.1 cm3 g-1, as expected for SBA-15 silica5 prepared under the adopted conditions, were calculated from N2 physisorption measurements. It should be also pointed out that the adsorptiondesorption isotherm in Figure 2a shows a steep N2 uptake at low relative pressures that indicates the presence of microporosity in the SBA-15 matrix. This was maybe due to intrawall micropores. Indeed, micropores may form as a result of calcination of the template, the hydrophobic EO tails of which had partially penetrated the silica walls during preparation.29,30 However, as estimated by the t-plot (not shown), the contribution of microporosity is very limited, the micropore volume being 0.098 cm3 g-1 only. The structural and textural data obtained by TEM, XRD, and N2 physisorption investigations indicate a high degree of hexagonal mesoscopic organization and are in agreement with previous results.5 3.2. Lipase Immobilization on SBA-15 Mesoporous Silica. 3.2.1. Effect of pH on Loading of Physically Adsorbed Lipase. The immobilization process was carried out by dissolving the enzymatic preparation in four different buffer solutions at pH values from 5 to 8. The crude enzymatic preparation used in this work is a commercial powder containing carbohydrates, salts, and also proteins different from the lipase.31 Enzymatic loading is usually expressed as milligrams of immobilized enzyme per gram of support. Since in our case different proteins can be immobilized during the process, we chose to express the loading in terms of IA, “immobilized activity”25 per gram of support (µmol butyric acid/min/g of support),
IA )
Figure 1. TEM micrographs of the SBA-15 mesoporous silica imaged in side view (a) and top view (b) and the corresponding powder X-ray diffraction pattern (c).
the channels are periodically well-ordered in a twodimensional hexagonal structure. The XRD wide angle pattern (not reported) indicates the presence of the two haloes typical of amorphous SiO2. On the other hand, the small-angle XRD pattern, reported in Figure 1c, shows three well-resolved peaks that can be indexed as the (100), (110), and (200) reflections associated with an hexagonal symmetry. These results are in agreement with the presence of a two-dimensional hexagonal P6mm structure with a large unit-cell parameter and indicate that the structure detected in small domains by TEM observations is actually representative of a long-range order. Figure 2a reports the 77 K nitrogen adsorptiondesorption isotherm of the SBA-15 matrix. The isotherm can be classified as type IV and exhibits an H1-type
Ai - Ar msupp
(1)
where Ai is the initial activity (µmol butyric acid/min) of the immobilizing buffer solution, Ar is the residual activity (µmol butyric acid/min) measured in the immobilizing buffer solution, msupp is the mass (grams) of SBA-15 support. This way to express the loading is proportional to the amount of active lipase effectively immobilized. The activity lipase assay was the tributyrin hydrolysis at pH 7. Figure 3 shows the variation of lipase loading on SBA15 versus pH: it increases from 1301 µmol min-1 g-1 at pH 5, to 1649 µmol min-1g-1 at pH 6; it decreases to 990 µmol min-1 g-1 at pH 7 and remains constant at pH 8 (1020 µmol min-1 g-1). Takahashi et al.12 found that horseradish peroxidase and subtilisin were not adsorbed significantly on SBA-15, (28) Sing, K. S. W.; Everett, D. H.; Haul, R. A. W.; Moscou, L.; Pierotti, R. A.; Rouque´rol, J.; Siemieniewska, T. Pure Appl. Chem. 1985, 57, 603. (29) Vort, P. V. D.; Ravikovitch, P. I.; Jong, K. P. D.; Benjelloun, M.; Bavel, E. V.; Janssen, A. H.; Neimark, A. V.; Weckhuysen, B. M.; Vansant, E. F. J. Phys. Chem. B 2002, 106, 5873. (30) Kruk, M.; Jaroniec, M.; Ko, C.; Ryoo, R. Chem. Mater. 2000, 12, 1961. (31) Salis, A.; Sanjust, E.; Monduzzi, M.; Solinas, V. Biocat. Biotransf. In press.
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Figure 2. N2 adsorption/desorption isotherms (a) of SBA-15 silica and corresponding pore size distribution (b) as obtained by the Dollimore-Heal calculation method.
Figure 3. Variation of IA (immobilized activity) versus pH of the buffer solution used during the immobilization process according to eq 1.
while they were strongly adsorbed on FSM-16 and MCM41 mesoporous silica. Moreover, they found that the amount of adsorbed horseradish peroxidase was strongly dependent on pH for FSM-16 and practically independent of pH for SBA-15.11 The main difference among these materials is in the templating surfactant used for the synthesis: a nonionic for SBA-15 and a cationic for both FSM-16 and MCM-41. Takahashi et al.12 explained their results by suggesting that the cationic surfactant may lead to an enhancement of the anionic potential of the silanol groups of the silica surface. This sounds a bit surprising upon considering that these are pure silica materials, they have the same hexagonal array of the channels, and the surfactants should have been fully removed by calcination at high temperature (823 K). Results in Figure 3 show a clear dependence of enzyme loading on pH, with a maximum at pH 6. Since the isoelectric point of silica occurs at pH ∼2,16 at higher pH values the silica surface is negatively charged and can
interact with a positively charged surface of proteins. Upon increasing the pH, also the surface of the protein becomes progressively negatively charged and therefore a repulsive interaction with the silica support is expected. Thus a decrease of enzyme loading with increasing pH should be expected, because of the progressive neutralization of positive charges at the protein surface. This suggestion agrees with the observation that proteins show maximal adsorption when pH is near pI (isoelectric point), as reported by Norde.32 Moreover, it should be remarked that for a commercial preparation of M. javanicus lipase, two active bands with pI ranged between 5.9 and 6.5 were determined by Bjurlin et al. in a recent investigation on the composition and activity of commercial lipases.33 This agrees and further supports the findings shown in Figure 3. It should be recalled that lipases have a great affinity toward hydrophobic surfaces, as recently shown for M. javanicus lipase adsorbed by a hydrophobic porous polypropylene support.34 In that case the immobilized preparation was successfully used for biocatalysis in a solvent-free system. Also in this case, a combination of electrostatic and van der Waals interactions might be responsible for the physical adsorption of M. javanicus lipase on SBA-15 mesoporous silica. A maximum loading occurring at pH 6 may be also the cumulative result of many different interactions due to the presence of other materials in the commercial lipase preparation. 3.2.2. Effect of pH on Activity of Physically Adsorbed Lipase. The pH of the buffer of the lipase solution affects not only the loading but also the activity of the immobilized biocatalyst significantly. Figure 4 shows the hydrolytic activity as a function of the pH value of the immobilization solution. (32) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. (33) Bjurlin, M. A.; Bloomer, S.; Hass, M. J. J. Am. Oil Chem. Soc. 2001, 78, 153. (34) Salis, A.; Sanjust, E.; Solinas, V.; Monduzzi, M. J. Mol. Catal. B 2003, 24-25, 75.
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Figure 5. Dynamic evolution of adsorption of M. javanicus lipase on SBA-15 mesoporous silica. Scheme 1. Lipase Immobilization on SBA-15 by Chemical Adsorption Figure 4. Variation of hydrolytic activity of M. javanicus lipase on SBA-15 versus pH of the immobilization buffer solution. Table 1. Difference in Loading (LL) and Activity (LA) of M. javanicus Lipase on SBA-15 Mesoporous Support, Obtained at Different pH of the Buffer of the Immobilization Solutiona pH(i)
difference in loading LLpH(6) - LLpH(i) (%)
difference in activity LApH(6) - LApH(i) (%)
5 6 7 8
21.1 0 40.0 38.1
21.1 0 83.7 81.6
a Values at pH 6 are taken as 100 %. All experiments were carried out at 298 K.
The trend parallels qualitatively that of loading. Again pH 6 favors the maximum activity. Table 1 reports the variation of loading and activity data with respect to the highest values obtained at pH 6. A loading variation of 21%, obtained varying the pH of the buffer from 5 to 6, produces an equal variation of enzymatic activity. On the contrary, at pH 7, a decrease of 40% and of 83.7% in loading and in enzymatic activity respectively is obtained. Values of loading and activity at pH 8 are quite similar to those at pH 7. Clearly pH values in the range 5-6 address the amount of immobilized enzyme. pH values in the range 7-8 inhibit lipase activity for two reasons: a lower loading and a possible change of the enzyme conformation, due to an unfavorable charge distribution on the amino acid residues, that produces a further activity decrease. Therefore, pH 6 was chosen for the other steps of this work. 3.2.3. Dynamic Evolution of the Immobilization Process by Physical Adsorption. In the main, protein adsorption on porous materials includes the following four steps: (1) protein diffusion from the bulk to the external surface of the support, (2) protein diffusion inside the channels, (3) adsorption of the protein at the solid surface, and (4) structural rearrangements of the protein. The dynamic evolution of the immobilization process, by physical adsorption, was followed by measuring the residual activity of the enzymatic buffer solution (pH 6). Figure 5 shows that about 50% of the activity disappears from the solutionsas a result of lipase molecules adsorptionsin the first 5 min of the process. The lowest residual activity, about 23% of the initial activity, is reached after 3 h. After this time, it is not more convenient to continue the adsorption process. These data should be compared to those obtained in a previous work, where the same commercial lipase preparation (from M. javanicus) was immobilized on a porous
polypropylene.34 In that case, lipase immobilization reached the equilibrium after 5 h. However, a higher amount of lipase was immobilized since only 5% of the initial activity was measured in the immobilization solution at the end of the process. The differences in the adsorption rate and amount of adsorbed enzyme of the same enzymatic preparation under the same conditions (20 mM phosphate buffer solution at pH 6, T ) 298 K) may be related to a different adsorption mechanism, probably due to different intermolecular forces. It is remarkable that, although van der Waals interactions in the case of polypropylene support and both electrostatic and van der Waals interactions for the SBA-15 silica are to be expected, in both cases pH 6 seems to play a key role. 3.2.4. Lipase Immobilization by Chemical Adsorption. Immobilized enzymes, and especially lipases, can be used both in aqueous and in nonaqueous media. In organic media, a strong enzyme/support interaction is not required, because of enzyme insolubility. Thus, physical adsorption is an appropriate immobilization method. On the contrary, as observed by Yu et al.,13 enzyme leaching in water media, can be an important phenomenon. To avoid such undesired phenomenon, a stronger enzyme/ support interaction, such as chemical adsorption, should be used. Therefore, the SBA-15 support was chemically modified as reported in Scheme 1. In the first step, the reaction between the -OH groups of the SBA-15 and the 3-aminopropyltriethoxysilane was
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performed; then the product of this reaction was treated with glutardialdehyde. In the last step, the superficial amino groups of the lipase were made to react with the functionalized support to produce the chemically adsorbed SBA-15/lipase biocatalyst. For this biocatalyst, chemical characterizations and optimal loading conditions are still under investigation. However, some preliminary results related to the comparison of the enzymatic activity toward tributyrin and triolein assays of the three preparations (free lipase and physical and chemical adsorption) are here reported. 3.3. Comparison between Tributyrin and Triolein Assays. Although a direct comparison among the three different conditions (free lipase and physical and chemical adsorption) cannot be done since the amount of loading in the chemical immobilization process is not known, the comparison between tributyrin and triolein assay responses in the different conditions may be interesting, provided that the same preparation is used to perform both assays. Hence, the activity ratios (Atributyrin/Atriolein) toward tributyrin and triolein substrates were determined for M. javanicus lipase under the following conditions: free, physically adsorbed, and chemically adsorbed on SBA-15. Activity of free lipase measured by tributyrin assay is higher than that obtained with triolein (Atributyrin/Atriolein ) 4.1). After physical adsorption of lipase on SBA-15, the activity measured with the two substrates becomes equal (Atributyrin/Atriolein ) 0.99). Chemically adsorbed lipase displays again a higher activity when tributyrin is used as substrate (Atributyrin/Atriolein ) 72.5). Enzymes should display their maximum activity under free conditions. However, for a particular enzyme the value of the activity varies depending on the used substrate. Thus, enzymatic activity of water-dissolved M. javanicus lipase is higher when tributyrin assay is used instead of triolein assay. After immobilization by physical adsorption, there is a noticeable effect of the support on the enzymatic activity. The process seems to be controlled by the diffusion of the substrates (both tributyrin and triolein) inside channels of SBA-15. Substrates’ diffusion is likely to mask the lipase preference toward tributyrin; thus, similar enzymatic activities are measured. This holds, provided that immobilization had not induced structural modifications. More problematic is the interpretation of activities for chemically adsorbed lipase. The ratio between activities (Atributyrin/Atriolein) increases from 4.1, observed for the free lipase condition, to 72.5 for the chemical adsorption case. This may be due to the different dimensions of the two substrates. Figure 6 shows the optimized geometries in vacuo of tributyrin and triolein, obtained through molecular modeling. The largest sizes of unsolvated tributyrin and triolein molecules are 7.5 and 23.3 Å, respectively. Since triolein is larger than tributyrin, the lower activity toward triolein may be caused by the obstruction of the channels due to the chemical adsorption process. Clearly,
Salis et al.
Figure 6. Space-filling representations of tributyrin (a) and triolein (b) molecules. Molecular geometries were optimized in vacuo using the MMFF94 force field as implemented in Spartan 5.1.3.
physical adsorption induces channel obstruction to a lesser extent. These results indirectly confirm that enzyme molecules are immobilized mainly inside the SBA-15 channels. It can be suggested that the significant decrease of activity toward triolein substrate means that triolein molecules can hardly enter the channels of the chemically modified SBA-15 support. Therefore, triolein can be hydrolyzed only by a few lipase molecules immobilized on the external surface or at the entrance of the channels of the SBA-15 support. 4. Conclusions In this work, the performance of lipases, the most used enzymes in biotechnological applications, and SBA-15 mesoporous silica, a very interesting material as an enzyme support, was investigated. Two different immobilization techniques were used: physical and chemical adsorption. They can be used both in aqueous or nonaqueous media, although chemical immobilization is usually preferred in aqueous solvents, while physical immobilization is sufficient in organic solvents. Hydrolytic activity measurements indicated that the chemically immobilized biocatalyst acquires size-selectivity as deduced from a direct comparison between tributyrin and triolein assays. Acknowledgment. S.L. thanks Consorzio UNO (Oristano) for her Ph.D. scholarship. E.D. thanks MIUR (Italy) for the Visiting Professor position at Cagliari University within the “Rientro Cervelli” Project. CSGI (Firenze) is acknowledged for support. Dr F. Mocci is also thanked for precious help in molecular modeling. LA047225Y
