Active Biocatalysts Based on Pepsin Immobilized in Mesoporous SBA

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J. Phys. Chem. C 2008, 112, 18110–18116

Active Biocatalysts Based on Pepsin Immobilized in Mesoporous SBA-15 Haresh G. Manyar,† Enrica Gianotti, Yasuhiro Sakamoto,‡ Osamu Terasaki,‡ Salvatore Coluccia,† and Simonetta Tumbiolo*,†,‡ Dipartimento di Chimica IFM and NIS - Centre of Excellence, UniVersity of Torino, Via P. Giuria 7, 10125 Torino, Italy, and Department of Structural Chemistry, Stockholm UniVersity, Arrhenius Laboratoriet, SVante Arrhenius Va¨g 12, 10691 Stockholm, Sweden ReceiVed: March 19, 2008; ReVised Manuscript ReceiVed: September 12, 2008

Porcine pepsin was immobilized inside the SBA-15 mesoporous silica system through physical adsorption. A grafting step with 3-aminopropryltriethoxysilane (APTES) was performed to reduce the pore openings of the host material, in order to minimize the enzyme leaching. A detailed physical chemical characterization of hybrid materials was performed. The catalytic activity of the hybrid bioinorganic material, tested with two different substrates (hemoglobin and Z-L-glutamyl-L-tyrosine dipeptide), confirmed that pepsin was located inside the pore/channels of the silica material and that the grafting process did not affect the enzyme structure. The immobilized pepsin has maintained the necessary degree of freedom to fulfill its catalytic activity. The reusability of the so-called bioreactor was also investigated. 1. Introduction Since the past decade, great interest has been focused on the adsorption of enzymes on ordered porous solids (such as zeolites and mesoporous molecular sieves) due to their unique pore structures.1-8 The immobilization of enzymes on solid supports allows researchers to perform highly selective catalysis using hybrid materials that are chemically and mechanically robust and readily separated from reaction mixtures.9,10 Recently, progress in the synthesis of mesoporous silica materials, characterized by controlled morphology, regular array of pores, and high surface areas, along with their chemical stability, has made silica matrices highly attractive as supports for the adsorption and immobilization of biomolecules in a confined space of nanometrical dimensions.11 Indeed, the immobilization in a confined space reduces enzyme autolysis (in the case of protease enzymes) and more generally reduces protein aggregation, allowing a better separation of enzyme molecules adsorbed onto the silica and enhancing the enzyme stability.12 Enzymes can be immobilized by cross-linking, covalent attachment, entrapment, and physical adsorption; the latter method is the most suitable since it does not affect the nature of the enzyme and it is a simple experimental procedure.13,14 When physical adsorption is used, the NH2 and CdO groups of the enzyme interact with silanol groups of the silica support; nevertheless this interaction is not strong enough to prevent the enzyme leaching. A covalent bond between enzyme and silica can be introduced to decrease the leaching, but frequently this procedure involves a loss in enzyme activity. In fact, covalent techniques require several chemical steps that can affect the nature of the enzyme, and the covalent attachment can reduce the enzyme conformational changes essential to interacting with the substrate.9,11 Leaching can also be minimized if the pore diameter of the mesoporous support is close to the size of the enzyme, but hence diffusion of substrate and products could * To whom correspondence should be addressed. E-mail: simonetta. [email protected]. Fax: +39 011 670 7953. Tel: +39 011 670 7536. † University of Torino. ‡ Stockholm University.

become severely restricted.14 Furthermore, leaching can be reduced also by decreasing the size of the pore openings of the mesoporous supports by silylation,4,9 modification of pore walls with thiol moieties,8,9 functionalization with carboxylic acid groups,15,9 or in situ polymerization of pendant vinyl groups, following the enzyme immobilization.16 A recent review by Hartmann summarizes the efforts made to achieve both active and reusable enzymes immobilized on ordered mesoporous solids.11 The resulting inorganic-organic hybrid materials, with the pore openings of the mesoporous support modified following the uptake of biocatalyst inside the channels, can be considered and used as a bioreactor. In this work, porcine pepsin (a bulky molecule of 34 kDa and dimensions of 55 × 74 × 36 Å)17,18 was immobilized by physical adsorption within the mesoporous silica SBA-15. Among different mesoporous silicas, SBA-15 was chosen due to the large pore dimensions (ca. 70 Å) and hexagonal array of pores that enable it to host bulky molecules such as proteins. In addition, to reduce the leaching, the enzyme immobilization was followed by a silylation step. Pepsin is one of three principal protein-degrading or proteolytic enzymes in the digestive system, the other two being chymotrypsin and trypsin. During the process of digestion, these enzymes, each of which is particularly effective in severing links between particular types of amino acids, collaborate to break down dietary proteins to their components, i.e., peptides and amino acids, which can be readily absorbed by the intestinal lining.19 Aspartic peptidases belonging to this family exhibit optimal activity at an acidic pH and contain two active-site aspartate residues required for function.20 Among proteolytic enzymes, pepsin having good protein-degrading activity and stability finds immense application in cheese manufacture from milk.18,21,22 Recently, pepsin has been immobilized on various supports, such as modified alumina complex (designing a continuously stirred tank reactor for producing bioactive hydrolysate),23 chemically modified poly(methyl methacrylate) microspheres,24 and activated Sepharose-4B25 for affinity chromatography. Immobilization of pepsin on agarose beads26 for in vitro

10.1021/jp802420t CCC: $40.75  2008 American Chemical Society Published on Web 10/29/2008

Active Biocatalysts Based on Pepsin refolding and on chitosan beads19 for application in the food industry is also reported. To the best of our knowledge, there are no reports about physical adsorption of pepsin within mesoporous structures and there are only few reports on the chemisorption of pepsin on synthetic and natural polymers and on inorganic oxides.27-30 SBA-15 mesoporous silica has already been used for bioimmobilization of small molecules such as cytochrom c, lysozyme, papain, trypsin, horseradish peroxidase, pancreatic lipase, etc.31-33 During a catalytic reaction, the pore dimensions of SBA-15 material permit an easy access of reactants to the active sites of the enzyme, as well the outgoing of the products. In this paper, the synthesis and a detailed physicochemical characterization of the so-called bioreactor, toghether with its catalytic activity, is reported. 2. Experimental Section 2.1. Materials. Porcine pepsin (PEP) was purchased from Sigma-Aldrich and used without further purification. All reagents for the synthesis of mesoporous and hybrid materials were purchased from Sigma-Aldrich: TEOS (tetraethylorthoxysilicate, 98%), Pluronic P123 triblock copolymer (PEO20PPO70PEO20), APTES (3-aminopropryltriethoxysilane), hydrochloric acid, toluene, acetone of AR grade, potassium acetate and acetic acid. Two substrates (hemoglobin from bovine blood and Z-Lglutamyl-L-tyrosine (Z-Glu-Tyr) dipeptide) used to test the catalytic activity of the hybrid materials, and trichloroacetic acid (TCA) used to stop the catalytic reactions, were also purchased from Sigma-Aldrich. Deionized AQUATRON A4D (Laiss) water was used for all preparations. 2.2. Synthesis of Mesoporous Silica SBA-15. According to the literature, SBA-15 mesoporous material was synthesized using Pluronic P123 triblock copolymer as the structure directing agent (SDA) and TEOS as the silica source.8 The solid product was washed three times with deionized water, filtered, and dried at room temperature. The removal of organic template was achieved by calcination under nitrogen flow, from room temperature to 550 °C with a heating step of 1 °C/min, and a holding time of 6 h at 550 °C under oxygen flow. The cooling rate was 5 °C/min. 2.3. Immobilization of Pepsin in Mesoporous SBA-15. Pepsin was immobilized into calcined SBA-15 by physical adsorption. A series of experiments to evaluate the amount of adsorbed pepsin at different pH was performed. Pepsin is unstable at pH > 6 (isoelectric point: 1). Experimentally, 50 mg of SBA-15 were suspended in 10 mL of pepsin solutions (2 mg/mL), prepared by varying the pH (2.6, 3.6, 4.6, and 5.8) of potassium acetate buffer. To evaluate the optimal amount of pepsin that can be immobilized inside the mesoporous channels, 50 mg of SBA15 were suspended in 10 mL of a series of pepsin solutions at different concentration (0.5, 0.75, 1.0, 1.25, 1.50, and 2.0 mg/ mL). In addition, 50 mg of as-synthesized SBA-15 (containing SDA) was also suspended in 10 mL of pepsin solution (2 mg/ mL of pure pepsin in potassium acetate buffer, pH 3.6). The hybrid material, named PEP/SBA-15, used for structural and textural characterization, was prepared adding 300 mg of calcined silica material to 10 mL of pepsin solution (2 mg/mL of pure pepsin in potassium acetate buffer at pH 3.6). All the mixtures (SBA-15 in pepsin solution) were stirred at room temperature, then centrifuged for 10 min at 5000 rpm and filtered. The enzyme concentration in the supernatant was analyzed using the absorbance values at 280 nm (pepsin ε280 ) 1.2803 M-1 cm-1) collected by UV spectrophotometer and a

J. Phys. Chem. C, Vol. 112, No. 46, 2008 18111 mass balance was applied to calculate the amount of enzyme adsorbed on the SBA-15. The band observed at 280 nm corresponds to the HOMO f LUMO electron transition in the aromatic rings of tryptophan and tyrosin residuals of pepsin molecules. Leakage of adsorbed enzyme into solution was analyzed as follows: 50 mg of hybrid material was mixed with 10 mL of buffer solution and stirred for 1 h at 300 rpm at room temperature. After centrifugation (20 min at 6000 rpm) and filtration, the enzyme concentration in the supernatant was analyzed by UV spectrophotometer. The percentage of enzyme leached out from the host material was calculated by difference from these enzyme content data. 2.4. Encapsulation Procedure. To reduce the degree of enzyme leaching from the mesoporous silica material, after the enzyme immobilization, the SBA-15 pore openings have been partially reduced by silylation with APTES. This encapsulation procedure allows the enzyme molecules to be trapped within the pores but still permits the reactant and product molecules to diffuse in and out of the pores. Experimentally, 50 mg of PEP/SBA-15 were added to 4 mL of APTES solution and 10 mL of toluene. After stirring for 3 h at 35 °C, the mixture was filtered, washed with acetone, and dried. The hybrid material produced after encapsulation will be named PEP/SBA-15/ APTES. 2.5. Characterization Techniques. X-ray diffraction patterns of calcined SBA-15 and hybrid materials were obtained on a Phillips PANalytical “X’Pert Pro” with Cu KR radiation (40 mA and 45 kV). Scanning electron micrographies (SEM) were performed using a LEICA Stereoscan operating at an accelerating voltage of 15 kV. Samples were prepared by placing SBA-15 powder on a double-sided carbon adhesive tape mounted on a sample holder and then sputtered with a thin film of gold to minimize the charging effects. High resolution transmission electron micrographies (HRTEM) were performed with a JEOL JEM-3010 microscope operating at 300 kV (Cs ) 0.6 mm, point resolution 1.7 Å). Images were recorded with CCD camera (MultiScan model 794, Gatan, 1024 × 1024 pixels, pixel size 24 × 24 µm2). The powder samples were mixed in ethanol and then ultrasonicated for 10 min. A drop of the wet sample was placed on a copper grid and then allowed to dry for 10 min before TEM analysis. Specific surface area (SSA), total pore volume and average pore diameter were measured by N2 adsorption-desorption isotherms at 77 K using Micromeritics ASAP 2020. The pore size was calculated on the adsorption branch of the isotherms using Barrett-Joyner-Helenda (BJH) method and the SSA was calculated using the Brunauer-Emmett-Teller (BET) method. DR UV-vis spectra were collected by Perkin-Elmer (Lambda 19) spectrometer equipped with an integrating sphere attachment. Fourier transform infrared (FT-IR) spectra of self-supporting wafers of the samples were collected with a Bruker IFS88 spectrometer at a resolution of 4 cm-1. Samples were outgassed at room temperature to remove the physically adsorbed water before FT-IR analysis. 2.6. Catalytic Activity Tests and Reusability. The catalytic activity was determined by estimating the amount of acid-soluble tyrosine and tryptophan residues released by reaction of pepsin on a substrate. Pepsin digests the substrate and yields acid soluble products which are readily detected by their strong UV feature at 280 nm. In a typical catalytic experiment, 0.4 mL of free enzyme solution (2 mg/mL in acetate buffer, pH ) 3.6) or the required amount of hybrid material (containing approxi-

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Figure 2. SEM (left) and TEM (right) micrographies of pure calcined SBA-15.

Figure 1. XRD diffraction pattern of calcined SBA-15 (curve a) and PEP/SBA-15 (curve b).

mately 0.8 mg of pepsin) were added to two different substrates: 2.0 mL of Z-Glu-Tyr dipeptide solution (2 g/L in acetate buffer, pH ) 3.6) or to 2.0 mL of hemoglobin solution (10 g/L in acetate buffer, pH ) 3.6). The first set of measurements was performed adding 4.0 mL of TCA solution (5% w/v) to the mixtures of free enzyme (or hybrid) and substrate to stop the catalytic reaction. The absorbance of the supernatant at 280 nm (A280) was used as reference value corresponding to enzyme and substrate contributions for a time close to zero. The second set of measurements was performed adding 4.0 mL of TCA solution (5% w/v) to the mixtures (enzyme or hybrid and substrate) after stirring for 20 min. Samples were centrifuged and filtered. The rate of hydrolysis of denatured substrates was calculated. The activity of pepsin was estimated from the increase in UV A280 of the supernatant. One unit of enzyme produces a change in absorbance at 280 nm of 0.001 per minute. The equation used for determining the specific activity was: U/mg ) [(∆A/min) × 1000]/[menzdi], where, ∆A/min is the increase in absorbance per minute, menz is the amount of enzyme, and di is the dilution factor.34 The reusability of PEP/SBA-15/APTES was studied up to 6 catalytic cycles with Z-Glu-Tyr as substrate. After each reaction cycle, immobilized pepsin was separated by centrifugation at 5000 rpm for 5 min. The recycled PEP/SBA-15/APTES was reintroduced in the reaction medium and the enzyme activities were detected for each cycle. 3. Results and Discussion 3.1. Structural and Textural Characterization of Pure SBA-15 and Hybrid Material. Pure SBA-15 and PEP/SBA15 hybrid were characterized by X-ray powder diffraction, SEM, HRTEM, and volumetric analysis. Calcined SBA-15 (Figure 1, curve a) shows the typical XRD pattern of an ordered hexagonal network of mesopores with (10), (11), and (20) reflections. The presence of well resolved (11) and (20) peaks indicates that the calcined material, used for the preparation of the hybrid material, has a long-range order. The hexagonal XRD pattern was still clearly observed in the hybrid material (PEP/SBA-15), as all of the three main reflections were found (Figure 1, curve b), indicating that the physical adsorption of pepsin does not affect the framework integrity of the material. The SEM image reported in Figure 2 (left) shows typical staked hexagonal disks and the two-dimensional hexagonal p6mm symmetry of the silica material with uniform diameter of the channel-pores was confirmed by HRTEM analysis (Figure 2, right).

Figure 3. Nitrogen adsorption/desorption isotherms at 77 K of calcined SBA-15 (curve a) and PEP/SBA-15 (curve b); the inset shows the pore size distribution of calcined SBA-15 (curve a) and PEP/SBA-15 (curve b).

Nitrogen adsorption/desorption isotherms at 77 K of calcined SBA-15 and hybrid material (PEP/SBA-15) are reported in Figure 3. Both calcined SBA-15 and hybrid materials exhibit a type IV adsorption isotherms (Brunauer definition) with a H1type hysteresis loop indicative of cylindrical pore shape. The volume of adsorbed nitrogen increases increasing relative pressure with a sharp rise in adsorption (between the relative pressure in the range 0.6-0.8 p/p0 in SBA-15 isotherm; Figure 3, curve a), due to the capillary condensation within uniform mesopores. Due to the condensation in textural porosity, a hysteresis loop, at pressure above 1.0 p/p0, is observed. The same inflections are present in the isotherm of the hybrid material (Figure 3, curve b). The mean pore diameter and the pore volume decrease respectively from 67 Å and 0.46 cm3/g for calcined SBA-15 to 60 Å and 0.32 cm3/g for PEP/SBA-15 (Figure 3, inset). A decrease of SSA was also observed: from 648 m2/g for SBA-15 to 418 m2/g for the hybrid material. These results suggest that pepsin molecules can be confined within the SBA-15 pores/channels and not simply adsorbed on the external surface of the silica. The hybrid PEP/SBA-15 system was also characterized by DR UV-vis spectroscopy. PEP/SBA-15 (Figure 4, solid line) shows the typical band at 280 nm due to the HOMO f LUMO electron transition in the aromatic rings of tryptophan and tyrosin residuals. Same absorption was, in fact, found in the UV-vis spectrum mode of pepsin in buffer solution (inset of Figure 4), while the pure calcined SBA-15 (Figure 4, dashed line) has no signal in this region. 3.2. Effect of pH on the Adsorption Rate of Pepsin on SBA-15. The isotherms of pepsin adsorbed on SBA-15 at different pH solutions ranging from 2.6 to 5.8 are shown in Figure 5. Each isotherm shows a sharp initial rise and reaches a plateau after 1 h. The amount of pepsin adsorbed on SBA-15 increases from pH 2.6 to 3.6 and then decreases upon further increase in pH solution to 4.6 and 5.8. The maximum of

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Figure 6. Adsorption curves of the amount of adsorbed pepsin within SBA-15 vs contacting time. Figure 4. DR UV-vis spectra of pure calcined SBA-15 (curve a) and PEP/SBA-15 (curve b); the inset shows the UV-vis spectrum of pepsin in buffer solution.

Figure 5. Effect of pH on the adsorption rate of pepsin on SBA-15.

adsorption is reached at pH 3.6, and hence, the solution at this pH is the optimum condition to adsorb pepsin on mesoporous system. At pH 3.6, pepsin is negatively charged in that its isoelectric point (pI) is 1. The pI of SBA-15, reported from several papers, ranges between 2.735 and 3.7.36,37 Our working pH is at the limit of the pI range of the silica. In this condition, it is difficult to determinate the absolute charge of the SBA-15 surface. At pH 3.6, our experimental data have evidenced that pepsin molecules are adsorbed into the inner silica surface. Nevertheless, the protein adsorption behavior is not completely understood. It is suggested that the driving forces, contributing to the adsorption of proteins on silica supports, are electrostatic forces, hydrogen bonds and hydrophobic interactions.9,35,38 Due to the high complexity of protein molecules, it is difficult to clarify completely which force is predominant and a synergic effect of forces should be hypothesized. 3.3. Effect of Pepsin Concentration on the Adsorption Rate. Experiments involving the adsorption of pepsin into SBA15 at room temperature were conducted for a range of initial concentrations (Cp) of protein ranging from 0.5 to 2.0 mg/mL. As seen in Figure 6, adsorption curves of the amount of pepsin adsorbed within SBA-15 as a function of contacting time showed little differences. For all initial enzyme concentrations, the saturation level was very fast and it was attained after 12 min of adsorption. At this contacting time, the adsorbed amount of pepsin was 108.5 mg/g for an initial pepsin concentration of 1 mg/mL and 117.4 mg/g for an initial pepsin concentration of 2.0 mg/mL. These results indicate that the kinetic adsorption rate of the enzyme within SBA-15 is independent from Cp in the range of concentration under study, while it is strictly related to the diffusion process inside the mesopores. The immobilization of lysozime into SBA-15 systems with different morphologies has produced similar results.6

3.4. Interaction between Enzyme and Support. To strengthen the fact that pepsin was immobilized into the mesopores of SBA-15 and not only adsorbed on its external surface, a series of experiments using as-synthesized SBA-15 as host was performed. In the as-synthesized SBA-15, mesopores and channels are still full of triblock copolymer used as SDA and only the external surface of the support was then available to interact with the enzyme. The maximum amount of pepsin adsorbed on the as-synthesized SBA-15 was 15 mg/g, whereas using calcined SBA-15, 117.4 mg/g was achieved. This result strongly supports the assumption that the inner surface of mesoporous material plays a key role in the immobilization of guest molecules. To evaluate the interaction between the enzyme and the mesoporous material, 300 mg of SBA-15 were suspended and stirred in 6, 8, 10, 12, and 14 mL of 3.0 mg/mL of pepsin solution, then the absorbance of supernatant was measured and the equilibrium concentration and adsorption amount were calculated. By fitting the experimental data with the Langmuir equation,6,39 we have calculated the maximum equilibrium adsorption amount (am ) 179 mg/g) of protein adsorbed per unit weight, and the dissociation coefficient (Kd ) 0.48 mg/ mL) of pepsin into silica mesopores, which represents the strong affinity between the solute and the adsorbents. During our experiments, SBA-15 has shown high-capacity for pepsin: 169 mg/g was the maximum amount of enzyme adsorbed into the material by physisorption (pepsin solution of 3.0 mg/mL, time of contact: 48 h). From literature, the highest capacities of pepsin adsorbed on poly (methyl methacrylate)/acryldehyde40 and on zirconia,30 both achieved by covalent binding, were 82 and 23 mg/g, respectively. The high adsorption of pepsin into the SBA15 system is probably due to an easy diffusion both within the large pores of the silica and within the “bridges” between adjacent channels.41 While the large mesopores allow the bulky pepsin molecule to diffuse in the material producing electrostatic and hydrophobic interactions, the Si-OH and/or Si-O- groups present on the inner walls of silica channels facilitate the enzyme immobilization, making the in-pore adsorption the rate-determining step during the whole immobilization process. The interaction between pepsin and silica surface was studied by FT-IR spectroscopy. FTIR spectra of calcined SBA-15 (curve a) and PEP/SBA-15 (curve b) are reported in Figure 7. The inset reports the spectrum of pure pepsin in KBr. In the high frequency region, calcined SBA-15 (curve a) shows a narrow peak at 3745 cm-1, due to the stretching mode of free silanol groups, overlapped to a broad adsorption at ca. 3535 cm-1 due to silanols interacting via H-bond.42 In fact, the sample is simply outgassed at r.t. and a fraction of Si-OH groups is still H-bonded. After the physical adsorption of pepsin (curve b), the bands due to silanols almost completely disappeared and a new broad absorption at 3350 cm-1 due to N-H stretching mode of pepsin

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Figure 7. FTIR spectra of calcined SBA-15 (curve a) and PEP/SBA15 (curve b). Both samples are outgassed at room temperature. In the inset, the FTIR spectrum of pure pepsin in KBr is reported.

TABLE 1: Volumetric Analysis Data Obteined from Nitrogen Adsorption/Desorption Isotherms at 77 K sample

mean pore diameter (Å)

pore volume (cm3 g-1)

pure calcined SBA-15 PEP/SBA-15 PEP/SBA-15/APTES

67 60 54

0.46 0.32 0.24

is formed. In addition, weak signals in the 3050-2850 cm-1 are present due to C-H stretching mode of -CH2 groups. Similar bands were in fact found in the pure pepsin spectrum (Figure 7, inset). In the low frequency range, the bands at 1655 and 1525 cm-1, only present in the PEP/SBA-15 spectrum, are typical of enzyme and are assigned to the stretching vibration of amide I and amide II, respectively.43,44 3.5. Leaching. The desorption factor of the immobilized protein was examined by evaluating the amount leached after a period of 1 h of strong mixing of PEP/SBA-15 in buffer solution. The desorption was important and the leaching amount was ca. 20%. Since these immobilization experiments were performed at pH 3.6 and since physical adsorption was occurring, it appears that immobilization is not strong enough to prevent a dynamic equilibrium between a solution phase and solid phase supported enzyme. However, to be a good biocatalyst, the leaching has to be less. To avoid this drawback, encapsulation with APTES has been carried out. Grafting is a common method followed to perform surface modification by covalent linking of organosilane molecules with surface silanol groups, reducing the pore opening of mesoporous material. Consequently, the enzyme leaching can be prevented, without inhibiting access to the substrate and releasing of the products. After APTES grafting, the mean pore diameter decreases from 60 to 54 Å and the pore volume decreases from 0.32 to 0.24 cm3/g. These data, obtained from nitrogen adsorption/desorption isotherms at 77 K, are reported in Table 1. In the same table, data of pure calcined SBA-15 and PEP/SBA-15 are reported for comparison. FTIR spectroscopy was used to obtain information on the APTES interaction with the silica surface. FTIR spectra of calcined SBA-15 (curve a) and APTES grafted on calcined SBA-15 (curve b) are reported in Figure 8. Both samples were outgassed at r.t. to remove molecular water. Calcined SBA-15 shows, in the high frequency region (Figure 8, section A), a narrow peak at 3745 cm-1 due to the stretching mode of free Si-OH and a broad band at ca. 3535 cm-1 due to silanols interacting via H-bond. In the low frequency region (Figure 8,

section B), calcined SBA-15 shows only a main absorption at ca. 810 cm-1 due to the symmetric stretching modes of Si-O-Si bridges of the silica network.44 Upon grafting with APTES (Figure 8, curve b), the bands due to the stretching modes of silanols completely disappeared and new absorptions in the 3400-2500 and 1650-1400 cm-1 ranges appear. The bands at 3355 and 3295 cm-1 are assigned to the asymmetric and symmetric N-H stretching modes of -NH2 groups, the weak signal at 3170 cm-1 is due to the first overtone of the -NH2 bending mode (fundamental at 1595 cm-1). The signals at 2935 and 2865 cm-1 are attributed to the asymmetric and symmetric C-H stretching modes of -CH2 groups. The absence of signals due to the stretching mode of -CH3 groups suggested that APTES molecules undergo hydrolysis that involve all the three ethoxy groups and therefore APTES is grafted on the silica surface by three R-Si-O-(silica) links. In the low frequency region (Figure 8, section B, curve b), the band at 1595 cm-1 is due to the bending mode of -NH2 groups. Weak signals present in the 1480-1350 cm-1 are due to the asymmetric and symmetric bending mode of -CH2 groups and the band at 690 cm-1 is assigned to the asymmetric O-Si-C stretching mode.45 In Figure 9, the FTIR spectra of SBA-15 grafted with APTES (curve a) and pepsin physically adsorbed on SBA-15 and then grafted with APTES (curve b) are compared. All the bands due to APTES and pepsin are present in the spectrum of PEP/SBA15/APTES, meaning that the treatment with APTES does not affect pepsin molecules. A remarkable decrease of the enzyme leaching was observed upon encapsulation with APTES, passing from 20% in the PEP/ SBA-15 sample to 7.8% in the PEP/SBA-15/APTES system. This behavior indicates that the encapsulation procedure has been successfully performed. 3.6. Catalytic Activity Tests. The catalytic activities of the free enzyme in buffer solution, PEP/SBA-15, and PEP/SBA15/APTES were evaluated by testing peptic hydrolysis of a solution of hemoglobin (a bulky molecule of 68 kDa, 574 amino acid residues, and dimensions of 68 × 72 × 115 Å)46 and of a solution of the smaller dipeptide Z-Glu-Tyr. Due to its dimensions, hemoglobin is unable to go through the channels of the SBA-15 material and could block the pore opening. In Table 2, the values of catalytic activity of free pepsin, PEP/SBA-15 and PEP/SBA-15/APTES are reported. The rate of hydrolysis of denatured substrates was measured. One enzymatic unit (U/ mg) releases 0.001A280 as TCA soluble hydrolysis products per minutes, under specified conditions. Catalytic activity of plain SBA-15 was also tested and no evidence of hydrolysis was found. Estimating the catalytic activity of free pepsin at 100%, the activity of the hybrid materials toward the dipeptide is higher than 98%, and toward hemoglobin is lower than 22%. We hypothesize that only the molecules of pepsin leached out of the pores/channels and those located on the external surface of the silica have interacted with hemoglobin, a big substrate that cannot enter inside the mesopores. Instead, Z-Glu-Tyr, that is a small molecule, can enter inside the pore/channels and interact with pepsin, that can fulfill its total catalytic activity. These data, in agreement with leaching and volumetric analysis data, confirm that the major part of pepsin is located into the inner surface of SBA-15 and not merely on the external surface. The same catalytic results are obtained for samples grafted with APTES (Table 2). It means that the encapsulation process does not affect the enzyme structure and permit a good diffusion of the dipeptide substrate and the products inside the pores. Finally, the reusability of the bioreactor PEP/SBA-15/APTES was studied up to 6 catalytic cycles. The hybrid material retained

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Figure 8. FTIR spectra of calcined SBA-15 (curve a) and calcined SBA-15 grafted with APTES (curve b). Section A: high frequency region; section B: low frequency region. Both samples are outgassed at room temperature.

Figure 9. FTIR spectra of calcined SBA-15 grafted with APTES (curve a) and PEP/SBA-15/APTES (curve b). Both samples are outgassed at room temperature.

TABLE 2: Catalytic Activity Tests for Peptic Hydrolysis Z-Glu-Tyr

hemoglobin

sample

enzyme activity (U/mg)

relative enzyme activity %

enzyme activity (U/mg)

relative enzyme activity %

free pepsin PEP/SBA-15 PEP/SBA-15/APTES

19.2 18.7 18.4

100 98 96

32.0 6.9 2.8

100 22 9

the 95% of activity up to 4 cycles and then the activity decreased slowly, until 65% after 6 catalytic cycles. In fact, 8% of enzyme amount was leached out after each cycle, and up to 6 catalytic cycles the bioreactor contained about 50% of the initial enzyme amount, resulting in a decreased activity. This indicates that the immobilized pepsin is recyclable up to 4 cycles without any loss of activity and can be reused effectively. 4. Conclusions In the present paper, the immobilization of pepsin by physical adsorption into the mesoporous silica SBA-15 was achieved successfully. The results obtained by fitting the experimental data with the Langmuir equation suggest a strong interaction between enzyme and inorganic support. The SBA-15 structure was not affected by the procedure used for enzyme immobiliza-

tion as evidenced by the structural characterization (XRD and HRTEM), and inside the mesopores, the pepsin molecules retain a certain degree of freedom to fulfill its catalytic activity. Volumetric analysis of the hybrid material evidenced that pepsin is adsorbed inside the mesopores, this feeling is also supported by the data obtained by adsorbing pepsin on as-synthesized SBA-15, in which the pores were full of template and pepsin was adsorbed only on the external surface. The leaching drawback has been successfully decreased by grafting the hybrid material with APTES; this procedure reduce the pore opening of SBA-15 without inhibiting access to the substrate and releasing of the products. Two different substrate, hemoglobin and Z-Glu-Tyr, were used to test the catalytic activity of our biocatalyst. The higher activity was achieved using Z-Glu-Tyr, due to its small dimensions that can enter the mesopores. On the contrary, the low catalytic activity, reported by using hemoglobin as substrate, can be explained considering that hemoglobin dimensions are not compatible with the pore opening of the mesoporous SBA-15. Thus, this substrate can interact only with pepsin leached out or attached on the external surface of the support. This behavior confirms that pepsin was chiefly adsorbed inside the mesopores. Our heterogeneous bioreactor produced by immobilization of pepsin inside the SBA-15 has shown high catalytic activity similar to the free enzyme and a good reusability. Acknowledgment. S.T. thanks the Boncompagni-Ludovisi Foundation for the financial support for the period spent in Sweden. H.G.M. thanks MIUR for finantial support. The authors acknowledge financial support by Regione Piemonte (Progetto CIPE 2005 No. D67 and Progetto NANOMAT, Docup 2000-2006, Linea 2.4a). The authors are also grateful to Compagnia di San Paolo for sponsorship to NIS - Centre of Excellence. References and Notes (1) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.; Schmitt, K. D.; Chu, C. T-W.; Olson, D. H.; Sheppard, E. W.; McCullen, S. B.; Higgins, J. B.; Schlenker, J. L. J. Am. Chem. Soc. 1992, 114, 10834. (2) Xia, Y.; Mokaya, R. Angew. Chem., Int. Ed. 2003, 42, 2639. (3) Fan, J.; Lei, J.; Wang, L.; Yu, C.; Tu, B.; Zhao, D. Chem. Commun. 2003, 2140. (4) Washmon-Kriel, L.; Jimenez, V. L.; Balkus, K. J. J. Mol. Catal. B: Enzym. 2000, 10, 453.

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