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 Vag 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. Agrafting step with 3-aminopropryltriethoxysilane (APTES) was performed to reduce the pore openings of thehost material, in order to minimize the enzyme leaching. A detailed physical chemical characterization ofhybrid materials was performed. The catalytic activity of the hybrid bioinorganic material, tested with twodifferent substrates (hemoglobin and Z-L-glutamyl-L-tyrosine dipeptide), confirmed that pepsin was locatedinside 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. Thereusability of the so-called bioreactor was also investigated.

1. Introduction

Since the past decade, great interest has been focused on theadsorption of enzymes on ordered porous solids (such as zeolitesand mesoporous molecular sieves) due to their unique porestructures.1-8 The immobilization of enzymes on solid supportsallows researchers to perform highly selective catalysis usinghybrid materials that are chemically and mechanically robustand readily separated from reaction mixtures.9,10

Recently, progress in the synthesis of mesoporous silicamaterials, characterized by controlled morphology, regular arrayof pores, and high surface areas, along with their chemicalstability, has made silica matrices highly attractive as supportsfor the adsorption and immobilization of biomolecules in aconfined space of nanometrical dimensions.11 Indeed, theimmobilization in a confined space reduces enzyme autolysis(in the case of protease enzymes) and more generally reducesprotein aggregation, allowing a better separation of enzymemolecules adsorbed onto the silica and enhancing the enzymestability.12

Enzymes can be immobilized by cross-linking, covalentattachment, entrapment, and physical adsorption; the lattermethod is the most suitable since it does not affect the natureof the enzyme and it is a simple experimental procedure.13,14

When physical adsorption is used, the NH2 and CdO groupsof the enzyme interact with silanol groups of the silica support;nevertheless this interaction is not strong enough to prevent theenzyme leaching. A covalent bond between enzyme and silicacan be introduced to decrease the leaching, but frequently thisprocedure involves a loss in enzyme activity. In fact, covalenttechniques require several chemical steps that can affect thenature of the enzyme, and the covalent attachment can reducethe enzyme conformational changes essential to interacting withthe substrate.9,11 Leaching can also be minimized if the porediameter of the mesoporous support is close to the size of theenzyme, but hence diffusion of substrate and products could

become severely restricted.14 Furthermore, leaching can bereduced also by decreasing the size of the pore openings of themesoporous supports by silylation,4,9 modification of pore wallswith thiol moieties,8,9 functionalization with carboxylic acidgroups,15,9 or in situ polymerization of pendant vinyl groups,following the enzyme immobilization.16

A recent review by Hartmann summarizes the efforts madeto achieve both active and reusable enzymes immobilized onordered mesoporous solids.11 The resulting inorganic-organichybrid materials, with the pore openings of the mesoporoussupport modified following the uptake of biocatalyst inside thechannels, can be considered and used as a bioreactor.

In this work, porcine pepsin (a bulky molecule of 34 kDaand dimensions of 55 × 74 × 36 Å)17,18 was immobilized byphysical adsorption within the mesoporous silica SBA-15.Among different mesoporous silicas, SBA-15 was chosen dueto the large pore dimensions (ca. 70 Å) and hexagonal array ofpores that enable it to host bulky molecules such as proteins.In addition, to reduce the leaching, the enzyme immobilizationwas followed by a silylation step.

Pepsin is one of three principal protein-degrading or pro-teolytic enzymes in the digestive system, the other two beingchymotrypsin and trypsin. During the process of digestion, theseenzymes, each of which is particularly effective in severing linksbetween particular types of amino acids, collaborate to breakdown dietary proteins to their components, i.e., peptides andamino acids, which can be readily absorbed by the intestinallining.19 Aspartic peptidases belonging to this family exhibitoptimal activity at an acidic pH and contain two active-siteaspartate residues required for function.20 Among proteolyticenzymes, pepsin having good protein-degrading activity andstability finds immense application in cheese manufacture frommilk.18,21,22

Recently, pepsin has been immobilized on various supports,such as modified alumina complex (designing a continuouslystirred 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

* To whom correspondence should be addressed. E-mail: simonetta.tumbiolo@unito.it. Fax: +39 011 670 7953. Tel: +39 011 670 7536.

† University of Torino.‡ Stockholm University.

J. Phys. Chem. C 2008, 112, 18110–1811618110

10.1021/jp802420t CCC: $40.75 2008 American Chemical SocietyPublished on Web 10/29/2008

refolding and on chitosan beads19 for application in the foodindustry is also reported. To the best of our knowledge, thereare no reports about physical adsorption of pepsin withinmesoporous structures and there are only few reports on thechemisorption of pepsin on synthetic and natural polymers andon inorganic oxides.27-30 SBA-15 mesoporous silica has alreadybeen used for bioimmobilization of small molecules such ascytochrom c, lysozyme, papain, trypsin, horseradish peroxidase,pancreatic lipase, etc.31-33 During a catalytic reaction, the poredimensions of SBA-15 material permit an easy access ofreactants to the active sites of the enzyme, as well the outgoingof the products.

In this paper, the synthesis and a detailed physicochemicalcharacterization of the so-called bioreactor, toghether with itscatalytic activity, is reported.

2. Experimental Section

2.1. Materials. Porcine pepsin (PEP) was purchased fromSigma-Aldrich and used without further purification. Allreagents for the synthesis of mesoporous and hybrid materialswere purchased from Sigma-Aldrich: TEOS (tetraethylorthoxy-silicate, 98%), Pluronic P123 triblock copolymer (PEO20PPO70-PEO20), APTES (3-aminopropryltriethoxysilane), hydrochloricacid, toluene, acetone of AR grade, potassium acetate and aceticacid. Two substrates (hemoglobin from bovine blood and Z-L-glutamyl-L-tyrosine (Z-Glu-Tyr) dipeptide) used to test thecatalytic activity of the hybrid materials, and trichloroacetic acid(TCA) used to stop the catalytic reactions, were also purchasedfrom Sigma-Aldrich. Deionized AQUATRON A4D (Laiss)water was used for all preparations.

2.2. Synthesis of Mesoporous Silica SBA-15. According tothe literature, SBA-15 mesoporous material was synthesizedusing Pluronic P123 triblock copolymer as the structure directingagent (SDA) and TEOS as the silica source.8 The solid productwas washed three times with deionized water, filtered, and driedat room temperature. The removal of organic template wasachieved by calcination under nitrogen flow, from roomtemperature to 550 °C with a heating step of 1 °C/min, and aholding time of 6 h at 550 °C under oxygen flow. The coolingrate was 5 °C/min.

2.3. Immobilization of Pepsin in Mesoporous SBA-15.Pepsin was immobilized into calcined SBA-15 by physicaladsorption. A series of experiments to evaluate the amount ofadsorbed pepsin at different pH was performed. Pepsin isunstable at pH > 6 (isoelectric point: 1). Experimentally, 50mg 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 beimmobilized inside the mesoporous channels, 50 mg of SBA-15 were suspended in 10 mL of a series of pepsin solutions atdifferent 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 (containingSDA) 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 structuraland textural characterization, was prepared adding 300 mg ofcalcined silica material to 10 mL of pepsin solution (2 mg/mLof pure pepsin in potassium acetate buffer at pH 3.6).

All the mixtures (SBA-15 in pepsin solution) were stirred atroom temperature, then centrifuged for 10 min at 5000 rpm andfiltered. The enzyme concentration in the supernatant wasanalyzed using the absorbance values at 280 nm (pepsin ε280 )1.2803 M-1 cm-1) collected by UV spectrophotometer and a

mass balance was applied to calculate the amount of enzymeadsorbed on the SBA-15. The band observed at 280 nmcorresponds to the HOMO f LUMO electron transition in thearomatic rings of tryptophan and tyrosin residuals of pepsinmolecules.

Leakage of adsorbed enzyme into solution was analyzed asfollows: 50 mg of hybrid material was mixed with 10 mL ofbuffer solution and stirred for 1 h at 300 rpm at roomtemperature. After centrifugation (20 min at 6000 rpm) andfiltration, the enzyme concentration in the supernatant wasanalyzed by UV spectrophotometer. The percentage of enzymeleached out from the host material was calculated by differencefrom these enzyme content data.

2.4. Encapsulation Procedure. To reduce the degree ofenzyme leaching from the mesoporous silica material, after theenzyme immobilization, the SBA-15 pore openings have beenpartially reduced by silylation with APTES. This encapsulationprocedure allows the enzyme molecules to be trapped withinthe pores but still permits the reactant and product moleculesto diffuse in and out of the pores. Experimentally, 50 mg ofPEP/SBA-15 were added to 4 mL of APTES solution and 10mL of toluene. After stirring for 3 h at 35 °C, the mixture wasfiltered, washed with acetone, and dried. The hybrid materialproduced after encapsulation will be named PEP/SBA-15/APTES.

2.5. Characterization Techniques. X-ray diffraction patternsof calcined SBA-15 and hybrid materials were obtained on aPhillips PANalytical “X’Pert Pro” with Cu KR radiation (40mA and 45 kV).

Scanning electron micrographies (SEM) were performedusing a LEICA Stereoscan operating at an accelerating voltageof 15 kV. Samples were prepared by placing SBA-15 powderon a double-sided carbon adhesive tape mounted on a sampleholder and then sputtered with a thin film of gold to minimizethe charging effects.

High resolution transmission electron micrographies (HR-TEM) were performed with a JEOL JEM-3010 microscopeoperating 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). Thepowder samples were mixed in ethanol and then ultrasonicatedfor 10 min. A drop of the wet sample was placed on a coppergrid and then allowed to dry for 10 min before TEM analysis.

Specific surface area (SSA), total pore volume and averagepore diameter were measured by N2 adsorption-desorptionisotherms at 77 K using Micromeritics ASAP 2020. The poresize was calculated on the adsorption branch of the isothermsusing Barrett-Joyner-Helenda (BJH) method and the SSA wascalculated using the Brunauer-Emmett-Teller (BET) method.

DR UV-vis spectra were collected by Perkin-Elmer (Lambda19) spectrometer equipped with an integrating sphere attachment.

Fourier transform infrared (FT-IR) spectra of self-supportingwafers of the samples were collected with a Bruker IFS88spectrometer at a resolution of 4 cm-1. Samples were outgassedat room temperature to remove the physically adsorbed waterbefore FT-IR analysis.

2.6. Catalytic Activity Tests and Reusability. The catalyticactivity was determined by estimating the amount of acid-solubletyrosine and tryptophan residues released by reaction of pepsinon a substrate. Pepsin digests the substrate and yields acidsoluble products which are readily detected by their strong UVfeature at 280 nm. In a typical catalytic experiment, 0.4 mL offree enzyme solution (2 mg/mL in acetate buffer, pH ) 3.6) orthe required amount of hybrid material (containing approxi-

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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 inacetate buffer, pH ) 3.6). The first set of measurements wasperformed adding 4.0 mL of TCA solution (5% w/v) to themixtures of free enzyme (or hybrid) and substrate to stop thecatalytic reaction. The absorbance of the supernatant at 280 nm(A280) was used as reference value corresponding to enzymeand substrate contributions for a time close to zero. The secondset of measurements was performed adding 4.0 mL of TCAsolution (5% w/v) to the mixtures (enzyme or hybrid andsubstrate) after stirring for 20 min. Samples were centrifugedand filtered. The rate of hydrolysis of denatured substrates wascalculated. The activity of pepsin was estimated from theincrease in UV A280 of the supernatant. One unit of enzymeproduces 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 theincrease 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 6catalytic cycles with Z-Glu-Tyr as substrate. After each reactioncycle, immobilized pepsin was separated by centrifugation at5000 rpm for 5 min. The recycled PEP/SBA-15/APTES wasreintroduced in the reaction medium and the enzyme activitieswere detected for each cycle.

3. Results and Discussion

3.1. Structural and Textural Characterization of PureSBA-15 and Hybrid Material. Pure SBA-15 and PEP/SBA-15 hybrid were characterized by X-ray powder diffraction, SEM,HRTEM, and volumetric analysis.

Calcined SBA-15 (Figure 1, curve a) shows the typical XRDpattern 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 thepreparation of the hybrid material, has a long-range order. Thehexagonal XRD pattern was still clearly observed in the hybridmaterial (PEP/SBA-15), as all of the three main reflections werefound (Figure 1, curve b), indicating that the physical adsorptionof pepsin does not affect the framework integrity of the material.The SEM image reported in Figure 2 (left) shows typical stakedhexagonal disks and the two-dimensional hexagonal p6mmsymmetry of the silica material with uniform diameter of thechannel-pores was confirmed by HRTEM analysis (Figure 2,right).

Nitrogen adsorption/desorption isotherms at 77 K of calcinedSBA-15 and hybrid material (PEP/SBA-15) are reported inFigure 3. Both calcined SBA-15 and hybrid materials exhibit atype IV adsorption isotherms (Brunauer definition) with a H1-type hysteresis loop indicative of cylindrical pore shape. Thevolume of adsorbed nitrogen increases increasing relativepressure with a sharp rise in adsorption (between the relativepressure in the range 0.6-0.8 p/p0 in SBA-15 isotherm; Fig-ure 3, curve a), due to the capillary condensation within uniformmesopores. Due to the condensation in textural porosity, ahysteresis loop, at pressure above 1.0 p/p0, is observed. Thesame inflections are present in the isotherm of the hybridmaterial (Figure 3, curve b). The mean pore diameter and thepore volume decrease respectively from 67 Å and 0.46 cm3/gfor 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: from648 m2/g for SBA-15 to 418 m2/g for the hybrid material. Theseresults suggest that pepsin molecules can be confined withinthe SBA-15 pores/channels and not simply adsorbed on theexternal surface of the silica.

The hybrid PEP/SBA-15 system was also characterized byDR UV-vis spectroscopy. PEP/SBA-15 (Figure 4, solid line)shows the typical band at 280 nm due to the HOMOf LUMOelectron transition in the aromatic rings of tryptophan and tyrosinresiduals. Same absorption was, in fact, found in the UV-visspectrum mode of pepsin in buffer solution (inset of Figure 4),while the pure calcined SBA-15 (Figure 4, dashed line) has nosignal in this region.

3.2. Effect of pH on the Adsorption Rate of Pepsin onSBA-15. The isotherms of pepsin adsorbed on SBA-15 atdifferent pH solutions ranging from 2.6 to 5.8 are shown inFigure 5. Each isotherm shows a sharp initial rise and reachesa plateau after 1 h. The amount of pepsin adsorbed on SBA-15increases from pH 2.6 to 3.6 and then decreases upon furtherincrease in pH solution to 4.6 and 5.8. The maximum of

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

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

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

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adsorption is reached at pH 3.6, and hence, the solution at thispH is the optimum condition to adsorb pepsin on mesoporoussystem.

At pH 3.6, pepsin is negatively charged in that its isoelectricpoint (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 limitof the pI range of the silica. In this condition, it is difficult todeterminate the absolute charge of the SBA-15 surface. At pH3.6, our experimental data have evidenced that pepsin moleculesare adsorbed into the inner silica surface. Nevertheless, theprotein adsorption behavior is not completely understood. It issuggested that the driving forces, contributing to the adsorptionof proteins on silica supports, are electrostatic forces, hydrogenbonds and hydrophobic interactions.9,35,38 Due to the highcomplexity of protein molecules, it is difficult to clarify com-pletely which force is predominant and a synergic effect offorces should be hypothesized.

3.3. Effect of Pepsin Concentration on the AdsorptionRate. Experiments involving the adsorption of pepsin into SBA-15 at room temperature were conducted for a range of initialconcentrations (Cp) of protein ranging from 0.5 to 2.0 mg/mL.As seen in Figure 6, adsorption curves of the amount of pepsinadsorbed within SBA-15 as a function of contacting time showedlittle differences. For all initial enzyme concentrations, thesaturation level was very fast and it was attained after 12 minof adsorption. At this contacting time, the adsorbed amount ofpepsin was 108.5 mg/g for an initial pepsin concentration of 1mg/mL and 117.4 mg/g for an initial pepsin concentration of2.0 mg/mL. These results indicate that the kinetic adsorptionrate of the enzyme within SBA-15 is independent from Cp inthe range of concentration under study, while it is strictly relatedto the diffusion process inside the mesopores. The immobiliza-tion of lysozime into SBA-15 systems with different morphol-ogies has produced similar results.6

3.4. InteractionbetweenEnzymeandSupport.Tostrengthenthe fact that pepsin was immobilized into the mesopores ofSBA-15 and not only adsorbed on its external surface, a seriesof experiments using as-synthesized SBA-15 as host wasperformed. In the as-synthesized SBA-15, mesopores andchannels are still full of triblock copolymer used as SDAand only the external surface of the support was then availableto interact with the enzyme. The maximum amount of pepsinadsorbed 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 innersurface of mesoporous material plays a key role in theimmobilization of guest molecules.

To evaluate the interaction between the enzyme and themesoporous material, 300 mg of SBA-15 were suspended andstirred in 6, 8, 10, 12, and 14 mL of 3.0 mg/mL of pepsinsolution, then the absorbance of supernatant was measured andthe equilibrium concentration and adsorption amount werecalculated. By fitting the experimental data with the Langmuirequation,6,39 we have calculated the maximum equilibriumadsorption amount (am ) 179 mg/g) of protein adsorbed perunit weight, and the dissociation coefficient (Kd ) 0.48 mg/mL) of pepsin into silica mesopores, which represents the strongaffinity between the solute and the adsorbents. During ourexperiments, SBA-15 has shown high-capacity for pepsin: 169mg/g was the maximum amount of enzyme adsorbed into thematerial by physisorption (pepsin solution of 3.0 mg/mL, timeof contact: 48 h). From literature, the highest capacities of pepsinadsorbed on poly (methyl methacrylate)/acryldehyde40 and onzirconia,30 both achieved by covalent binding, were 82 and 23mg/g, respectively. The high adsorption of pepsin into the SBA-15 system is probably due to an easy diffusion both within thelarge pores of the silica and within the “bridges” betweenadjacent channels.41 While the large mesopores allow the bulkypepsin molecule to diffuse in the material producing electrostaticand hydrophobic interactions, the Si-OH and/or Si-O- groupspresent on the inner walls of silica channels facilitate the enzymeimmobilization, making the in-pore adsorption the rate-deter-mining step during the whole immobilization process.

The interaction between pepsin and silica surface was studiedby FT-IR spectroscopy. FTIR spectra of calcined SBA-15 (curvea) and PEP/SBA-15 (curve b) are reported in Figure 7. Theinset reports the spectrum of pure pepsin in KBr. In the highfrequency region, calcined SBA-15 (curve a) shows a narrowpeak at 3745 cm-1, due to the stretching mode of free silanolgroups, overlapped to a broad adsorption at ca. 3535 cm-1 dueto silanols interacting via H-bond.42 In fact, the sample is simplyoutgassed at r.t. and a fraction of Si-OH groups is still H-bonded.After the physical adsorption of pepsin (curve b), the bandsdue to silanols almost completely disappeared and a new broadabsorption at 3350 cm-1 due to N-H stretching mode of pepsin

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 ofpepsin in buffer solution.

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

Figure 6. Adsorption curves of the amount of adsorbed pepsin withinSBA-15 vs contacting time.

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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 1655and 1525 cm-1, only present in the PEP/SBA-15 spectrum, aretypical of enzyme and are assigned to the stretching vibrationof amide I and amide II, respectively.43,44

3.5. Leaching. The desorption factor of the immobilizedprotein was examined by evaluating the amount leached after aperiod 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 performedat pH 3.6 and since physical adsorption was occurring, it appearsthat immobilization is not strong enough to prevent a dynamicequilibrium between a solution phase and solid phase supportedenzyme. However, to be a good biocatalyst, the leaching has tobe less. To avoid this drawback, encapsulation with APTEShas been carried out. Grafting is a common method followedto perform surface modification by covalent linking of orga-nosilane molecules with surface silanol groups, reducing thepore opening of mesoporous material. Consequently, the enzymeleaching can be prevented, without inhibiting access to thesubstrate and releasing of the products.

After APTES grafting, the mean pore diameter decreases from60 to 54 Å and the pore volume decreases from 0.32 to 0.24cm3/g. These data, obtained from nitrogen adsorption/desorptionisotherms at 77 K, are reported in Table 1. In the same table,data of pure calcined SBA-15 and PEP/SBA-15 are reportedfor comparison.

FTIR spectroscopy was used to obtain information on theAPTES interaction with the silica surface. FTIR spectra ofcalcined SBA-15 (curve a) and APTES grafted on calcinedSBA-15 (curve b) are reported in Figure 8. Both samples wereoutgassed at r.t. to remove molecular water. Calcined SBA-15shows, in the high frequency region (Figure 8, section A), anarrow peak at 3745 cm-1 due to the stretching mode of freeSi-OH and a broad band at ca. 3535 cm-1 due to silanolsinteracting via H-bond. In the low frequency region (Figure 8,

section B), calcined SBA-15 shows only a main absorption atca. 810 cm-1 due to the symmetric stretching modes ofSi-O-Si bridges of the silica network.44 Upon grafting withAPTES (Figure 8, curve b), the bands due to the stretchingmodes of silanols completely disappeared and new absorptionsin the 3400-2500 and 1650-1400 cm-1 ranges appear. Thebands at 3355 and 3295 cm-1 are assigned to the asymmetricand symmetric N-H stretching modes of -NH2 groups, theweak signal at 3170 cm-1 is due to the first overtone of the-NH2 bending mode (fundamental at 1595 cm-1). The signalsat 2935 and 2865 cm-1 are attributed to the asymmetric andsymmetric C-H stretching modes of -CH2 groups. The absenceof signals due to the stretching mode of -CH3 groups suggestedthat APTES molecules undergo hydrolysis that involve all thethree ethoxy groups and therefore APTES is grafted on the silicasurface by three R-Si-O-(silica) links. In the low frequencyregion (Figure 8, section B, curve b), the band at 1595 cm-1 isdue to the bending mode of -NH2 groups. Weak signals presentin the 1480-1350 cm-1 are due to the asymmetric andsymmetric bending mode of -CH2 groups and the band at 690cm-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 thengrafted with APTES (curve b) are compared. All the bands dueto APTES and pepsin are present in the spectrum of PEP/SBA-15/APTES, meaning that the treatment with APTES does notaffect pepsin molecules.

A remarkable decrease of the enzyme leaching was observedupon 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 hasbeen successfully performed.

3.6. Catalytic Activity Tests. The catalytic activities of thefree enzyme in buffer solution, PEP/SBA-15, and PEP/SBA-15/APTES were evaluated by testing peptic hydrolysis of asolution of hemoglobin (a bulky molecule of 68 kDa, 574 aminoacid residues, and dimensions of 68 × 72 × 115 Å)46 and of asolution of the smaller dipeptide Z-Glu-Tyr. Due to its dimen-sions, hemoglobin is unable to go through the channels of theSBA-15 material and could block the pore opening. In Table2, the values of catalytic activity of free pepsin, PEP/SBA-15and PEP/SBA-15/APTES are reported. The rate of hydrolysisof denatured substrates was measured. One enzymatic unit (U/mg) releases 0.001A280 as TCA soluble hydrolysis products perminutes, under specified conditions. Catalytic activity of plainSBA-15 was also tested and no evidence of hydrolysis wasfound. Estimating the catalytic activity of free pepsin at 100%,the activity of the hybrid materials toward the dipeptide is higherthan 98%, and toward hemoglobin is lower than 22%. Wehypothesize that only the molecules of pepsin leached out ofthe pores/channels and those located on the external surface ofthe silica have interacted with hemoglobin, a big substrate thatcannot enter inside the mesopores. Instead, Z-Glu-Tyr, that isa small molecule, can enter inside the pore/channels and interactwith pepsin, that can fulfill its total catalytic activity. These data,in agreement with leaching and volumetric analysis data, confirmthat the major part of pepsin is located into the inner surface ofSBA-15 and not merely on the external surface. The samecatalytic results are obtained for samples grafted with APTES(Table 2). It means that the encapsulation process does not affectthe enzyme structure and permit a good diffusion of thedipeptide substrate and the products inside the pores.

Finally, the reusability of the bioreactor PEP/SBA-15/APTESwas studied up to 6 catalytic cycles. The hybrid material retained

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

TABLE 1: Volumetric Analysis Data Obteined fromNitrogen Adsorption/Desorption Isotherms at 77 K

samplemean pore

diameter (Å)pore volume

(cm3 g-1)

pure calcined SBA-15 67 0.46PEP/SBA-15 60 0.32PEP/SBA-15/APTES 54 0.24

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the 95% of activity up to 4 cycles and then the activity decreasedslowly, until 65% after 6 catalytic cycles. In fact, 8% of enzymeamount was leached out after each cycle, and up to 6 catalyticcycles the bioreactor contained about 50% of the initial enzymeamount, resulting in a decreased activity. This indicates thatthe immobilized pepsin is recyclable up to 4 cycles without anyloss of activity and can be reused effectively.

4. Conclusions

In the present paper, the immobilization of pepsin by physicaladsorption into the mesoporous silica SBA-15 was achievedsuccessfully. The results obtained by fitting the experimentaldata with the Langmuir equation suggest a strong interactionbetween enzyme and inorganic support. The SBA-15 structurewas not affected by the procedure used for enzyme immobiliza-

tion as evidenced by the structural characterization (XRD andHRTEM), and inside the mesopores, the pepsin molecules retaina certain degree of freedom to fulfill its catalytic activity.Volumetric analysis of the hybrid material evidenced that pepsinis adsorbed inside the mesopores, this feeling is also supportedby the data obtained by adsorbing pepsin on as-synthesizedSBA-15, in which the pores were full of template and pepsinwas adsorbed only on the external surface. The leachingdrawback has been successfully decreased by grafting the hybridmaterial with APTES; this procedure reduce the pore openingof SBA-15 without inhibiting access to the substrate andreleasing of the products. Two different substrate, hemoglobinand Z-Glu-Tyr, were used to test the catalytic activity of ourbiocatalyst. The higher activity was achieved using Z-Glu-Tyr,due to its small dimensions that can enter the mesopores. Onthe contrary, the low catalytic activity, reported by usinghemoglobin as substrate, can be explained considering thathemoglobin dimensions are not compatible with the poreopening of the mesoporous SBA-15. Thus, this substrate caninteract only with pepsin leached out or attached on the externalsurface of the support. This behavior confirms that pepsin waschiefly adsorbed inside the mesopores. Our heterogeneousbioreactor produced by immobilization of pepsin inside theSBA-15 has shown high catalytic activity similar to the freeenzyme and a good reusability.

Acknowledgment. S.T. thanks the Boncompagni-LudovisiFoundation for the financial support for the period spent inSweden. H.G.M. thanks MIUR for finantial support. The authorsacknowledge financial support by Regione Piemonte (ProgettoCIPE 2005 No. D67 and Progetto NANOMAT, Docup2000-2006, Linea 2.4a). The authors are also grateful toCompagnia di San Paolo for sponsorship to NIS - Centre ofExcellence.

References and Notes

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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 (curvea) and PEP/SBA-15/APTES (curve b). Both samples are outgassed atroom temperature.

TABLE 2: Catalytic Activity Tests for Peptic Hydrolysis

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Active Biocatalysts Based on Pepsin J. Phys. Chem. C, Vol. 112, No. 46, 2008 18115

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