Enhanced Activity and Stability of Lysozyme by Immobilization in the

Mar 7, 2014 - Department of Chemistry, National Taiwan University, Taipei, ... from Mesoporous Silica Film for Surface-Enhanced Raman Spectroscopy...
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Enhanced Activity and Stability of Lysozyme by Immobilization in the Matching Nanochannels of Mesoporous Silica Nanoparticles Kun-Che Kao,† Tien-Sung Lin,‡ and Chung-Yuan Mou*,† †

Department of Chemistry, National Taiwan University, Taipei, Taiwan 106 Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States

‡

S Supporting Information *

ABSTRACT: It is highly desirable to study the kinetics and spectroscopy of enzymes in a crowded and controllable microenvironment. In this work, we employ mesoporous silica of matching pore sizes to confine lysozyme in order to mimic enzyme in a crowded environment. The stability and activity of lysozyme immobilized in mesoporous silica nanoparticle (MSN) of various pore sizes were studied and correlated to spectroscopic data of the immobilized enzyme. By siteselective surface functionalization, we were able to avoid protein adsorbing on the external surfaces of MSNs and study specifically the protein immobilized in the nanochannels. Solution spectroscopic methods, CD and fluorescence, were used to study the secondary and tertiary structures of the immobilized enzyme because MSNs could be suspended very well in solution. To study the catalytic activity of lysozyme, we employed 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside as a substrate that was hydrolyzed and detected by fluorescence spectroscopy. 8-Anilino-1-naphthalenesulfonic acid was utilized as a fluorescence probe to characterize the protein-binding site. The conformation, thermal stability, and catalytic activity of lysozyme were sensitive to the curvature of the silica materials. The activity of the lysozyme immobilized in the 5.6 nm mesopores of MSNs was higher than those of native enzymes. The enhanced activity was attributed to subtle change in tertiary structure of lysozyme in the crowded microenvironment in the mesopores.

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INTRODUCTION Much effort has been devoted to the development of methods for enzyme immobilization to realize numerous advantages, including enhanced stability, recyclability, easy separation, and prevention of contamination in the product.1 In the past decade, for applications in biosensing and protein delivery, organic and inorganic nanoparticles have been used to immobilize protein.2 However, immobilization of protein on nanoparticles could change its conformations. Surface properties of nanoparticles, such as surface functionality, porosity, curvature, and heterogeneity, could strongly influence the adsorption of protein and its structural stability and catalytic activity.3−7 In fact, immobilization of enzyme often leads to lower catalytic activity. This is generally true for enzyme loading on nanoparticles or flat surfaces because the structure of enzyme often becomes distorted. For example, Vertegel et al. reported that the structure of lysozyme was highly distorted when supported on silica nanoparticles and gave lower activity compared to free enzyme in solution.8 On the other hand, enzyme molecules supported on the concave surface of pore of comparable size often give enhanced activity and stability, in systems such as reverse microemulsion9 and mesoporous silica.10−13 In particular, mesoporous silicas are gaining much attentions as immobilization supports for enzymes.14−17 Several research groups further studied the © 2014 American Chemical Society

physical chemistry of mesoporous silicas for encapsulating proteins.14,15,18,19 For the motivation of this work, mesoporous silica provides two more opportunities to advance the science of enzyme. First, it could provide one with a model for studying the crowding effect of enzymes.1,10,12,13 The intracellular environment is highly crowded with biopolymers; the total macromolecular concentrations could reach 400 g/L. Cellular crowding provides stability against unfolding while modulating its activity. Silica matrix has been shown to be a good system for studying the crowding effect on protein structure.10,20 The uniform, functionalizable, and easily accessible pores could further make the correlation between confining environment and catalytic activity of enzyme realizable. Second, MSNs, a nanoparticular form of mesoporous silica (∼100 nm in diameter) suspensions, are nearly transparent in buffer solutions, which make it possible to directly measure the structural changes of encapsulated protein by solution spectroscopy. The structural study of protein adsorbed within the nanochannels of mesoporous silica is still in its infancy because the bulk mesoporous silicas, several micrometers in size, will Received: November 16, 2013 Revised: February 15, 2014 Published: March 7, 2014 6734

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alized MSNs were centrifuged and washed with ethanol three times to remove residual reagents. To remove CTAB templates, MSNs were dispersed in 50 mL of HCl/ethanol (5 mg/mL) and stirred at 50 °C for 20 h. The final particles were washed with ethanol six times and stored in 99.5% of ethanol. Lysozyme Adsorption Isotherms and Desorption Procedure. Adsorption curves for lysozyme were performed at pH 7.4 (20 mM sodium phosphate buffer). Ten milligrams of silica materials was mixed with 20 mL of lysozyme solution with concentrations from 75 to 800 μg/mL. The solutions were shaken at room temperature (26 °C) for 4 h. Then, the lysozyme−silica mixtures were centrifuged at 13 000 rpm for 20 min, and the residual concentrations of lysozyme in the supernatants were measured by UV−vis spectrometer at 280 nm. For more accurate measurements, the concentrations of lysozyme were determined by the Bradford method (Bio-Rad). The amounts of adsorbed lysozyme on silica materials were calculated from the differences between the initial and supernatants’ concentrations. For desorption procedures, the centrifuged lysozyme−silica mixtures were resuspended in 20 mL of sodium acetate solutions (20 mM) at pH 3.2 for 4 h. Analysis of Lysozyme Secondary Structure by CD Spectra. Far-UV CD spectra were measured to determine secondary structures of lysozyme samples on the basis of characteristic α-helix absorption at 209 and 222 nm. The spectra were fitted and the secondary structure contents of protein were calculated using the CDSSTR method.26 Ten milligrams of silica materials was mixed with 20 mL of lysozyme solution (in 20 mM of sodium phosphate buffer at pH 7.4) with initial concentration of 75 μg/mL. After adsorption at pH 7.4 for 4 h, the mixtures were centrifuged and the lysozyme molecules on external surface were desorbed in 20 mL of sodium acetate solution (20 mM) at pH 3.2 for 4 h. Then, the lysozyme−silica mixtures were centrifuged and resuspended in 20 mL of sodium phosphate buffer (20 mM) at pH 7.4 for 2 h. It is noted that all of the lysozyme in these samples was adsorbed; no free lysozyme was detected in the supernatants. Blank buffer or silica material solutions were also measured and subtracted from the lysozyme sample spectra. The amount of residual α-helix was monitored at 222 nm using a heating rate of 2 °C/min from 20 to 100 °C with stirring. Activity Assay of Lysozyme. To measure the catalytic activity of lysozyme, 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside [4-MU-β-(GlcNAc)3] was used as a substrate. The substrate could be hydrolyzed by lysozyme and formed 7hydroxy-4-methylcoumarin (4-MU), which could be detected by fluorescence spectroscopy.23 The immobilization procedures and initial lysozyme concetrations were the same as for the CD analyses experiments. The lysozyme−silica mixtures were suspended in sodium phosphate buffer (20 mM) at pH 7.4 for 2 h before reaction. Then, in a vial, 5 mL of free or adsorbed lysozyme solutions (all samples set to be 100 μg/mL) were mixed with 5 mL of 4-MU-β-(GlcNAc)3 (0.06 mg/mL). Every 20 min, 1.5 mL of the reacted solution was centrifuged at 4 °C (for quenching the reaction), and the supernatants were taken to measure the fluorescent intensity of cleaved 4-MU (λex = 355 nm and λem = 460 nm). The initial rate of lysozyme samples producing 4-MU was calculated between 20 and 100 min. To study the thermal stability of lysozyme on MSNs or SSN, lysozyme samples were hydrothermally incubated at 70 °C for 1 h before the catalytic activity assay. ANS Binding Experiments. 1-Anilinonaphthalene-8-sulfonate (ANS) binding experiments were performed using a

either give strong scattering of light or rapidly precipitate from aqueous solution. In this work, lysozyme, which has 129 residues with dimensions (4.5 × 3.0 × 3.0 nm3) comparable to the pore diameters of MSNs, was chosen as a model protein. Previously, we reported that various pore sizes of MSNs (up to 6.0 nm) could be synthesized using decane as a pore-expanding reagent.21 Taking advantage of the controllable particle size at sub-100 nm, narrow pore size distributions, and well-ordered pore structures of MSNs, we thereby study lysozyme encapsulated within their nanochannels to examine the protein’s stability and reactivity by circular dichroism (CD) and fluorescence spectroscopy. CD spectroscopy is a powerful technique to characterize the structure of protein in solutions. Recently, CD has been applied to monitor the conformational changes of protein during the interaction with nanomaterials.8,22 Lysozyme adsorbed in different pore sizes of MSNs and on the surface of Stöber silica nanoparticle (SSN) will be examined. The conformation, stability, and catalytic activity of lysozyme were very sensitive to the surface curvature and the pore size of the silica materials, which was in agreement with the previously reported results of lysozyme adsorbed in SBA-15 (monitored by ATR-FTIR).23 In addition, we found that the superactivity of confined lysozyme could be attributed to the subtle change of tertiary structures studied by fluorescence spectroscopy. Upon understanding the nature of interactions between MSNs and proteins, we may design an enzymecontaining nanocomposite with better stability to achieve effective enzyme delivery and biomedical applications. In addition, by proper surface functionalization of MSNs, we designed a strategy to purposely exclude the protein adsorbed on the external surfaces of MSNs through electrostatic interactions so that we could load the lysozyme only in the nanochannels. The ensuing catalytic effect of lysozyme can be attributed only to the confined enzymes in the interior pores. MSN has recently gained much attention in its wide applications in nanomedicine because of its easy and designable interaction with biological cells.24 Properly designed MSN− enzyme complex could also be developed as carriers for enzyme therapy.25

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EXPERIMENTAL SECTION Synthesis and Functionalization of MSNs. Materials sources and characterizations are given in the Supporting Information. MSNs with tunable pore size (2.5−6 nm) were synthesized following our previous method using decane as pore-expanding reagent.21 We synthesized three different pore sizes of MSNs by adding different amounts of decane. First, 0.386 g of CTAB was dissolved in 160 g of 0.3 M NH3 solution at 50 °C. Then, 0, 0.3, and 1.2 mL of decane were dissolved in 15 mL of ethanol, respectively. After that, aqueous CTAB solution was mixed with the ethanol solutions of decane and formed oil-in-water (O/W) emulsions. The microemulsions were stirred at 50 °C for 12 h, and then 3.33 mL of TEOS/ ethanol [20% (v/v)] solution was added and the mixture was kept under stirring at 50 °C for 1 h. These solutions were then aged at 50 °C for 20 h. The as-synthesized products were filtered to remove side products. After that, MSN solutions were hydrothermally treated at 80 °C for 24 h. To attach (3aminopropyl)trimethoxysilane (APTS) only on the external surfaces of MSNs, 20 μL of APTS was added into the MSNs solutions before extracting CTAB to avoid APTS penetrating into the nanochannels of MSNs. Then, the APTS-function6735

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below 100 nm (Table 1). The hydrodynamic diameters determined by dynamical light scattering (DLS) were just slightly larger than the particle sizes from TEM images (Table 1 and Supporting Information, Figure S2), indicating that the colloidal MSNs were well-suspended without aggregation. Thus, the conformational changes of immobilized lysozyme could be directly monitored in buffer solution using CD spectroscopy. In addition, it is relatively easy to load a large amount of biomolecules in these sub-100 nm channels.31 Recently, Piras et al. showed that lysozyme can be imaged inside the nanochannels of SBA-15 using an immunogold staining method.32 The short, open, and unobstructed channels in MSN lead to high protein adsorption. For investigating the confining effect on the conformation of lysozyme in nanochannels, we synthesized functionalized MSNN of three pore sizes by adding different amounts of decane in the synthesis. These APTS-functionalized MSNs were denoted as MSN-N-2.5, MSN-N-4.5, and MSN-N-5.6 with BJH pore sizes of 2.5, 4.5, and 5.6 nm, respectively. We note here that the BJH method probably underestimated (by 10−30%) the pore size; the labeling X in MSN-N-X should be taken as nominal labeling. Nitrogen adsorption−desorption isotherms of these MSN-N samples all give typical type IV adsorption isotherms (Figure 2A). Capillary condensation steps occurred at P/P0 = 0.9−1.0 were attributed to the textural porosity (interparticle spacing) of MSNs. The physical parameters of MSN-Ns samples are summarized in Table 1. BET surface areas of MSNNs are all around 1000 m2/g and the total pore volume increased from 1.49 to 2.27 cm3/g as pores were expanded. The large pore size of MSN-N-5.6 showed much higher mesopore volume (1.53 cm3/g) than the small pore size of MSN-N-2.5 (0.91 cm3/g). As a result, the amount of lysozyme adsorbed on MSN-N-5.6 was also higher than that on others (see Figure 4). Powder X-ray diffraction patterns of MSN-N samples (Figure 2B) all exhibit excellent mesostructures with four Bragg reflection peaks of 2D-hexagonal (p6mm) structures, which are consistent with the TEM results. The driving force of protein adsorption would be the electrostatic interaction between silica and proteins. From the zeta potential pH titration curves, the isoelectric points (pI) of lysozyme and bared MSNs are around 10.0 and 4.0, respectively (Figure 3a,c). We note also that the pI = 4 of MSN has increased a little compared with that of SSN (pI = 3), which is probably due to esterification of surface silanol to Si−O−Et during ethanol extraction. Thus, positively charged lysozyme could be easily adsorbed on the negatively charged surface of silica in the pH range of 4.0−10.0. However, it has been reported that multilayer protein molecules could be adsorbed outside of the nanochannels of mesoporous silica materials via Coulombic attraction, especially for nanosized particles due to the large external surface areas.33 To minimize adsorption on the external surface, MSNs were first functionalized with APTS only on external surfaces before the surfactant templates are extracted (Supporting Information, Figure S3). Figure 3 shows the zeta potential titration curves of the MSN-N samples, which all give isoelectric points at around pH 6.0. At pH 7.4, MSNs are negatively charged and lysozyme molecules could be adsorbed both inside and outside the nanochannels (Scheme 1). However, when the MSN-N samples were suspended in a buffer solution with pH 3.2, their external surfaces became positively charged due to the presence of functionalized amino groups (Figure 3b,d,e). As a result, lysozyme on the external surface of MSN could be desorbed from APTS-functionalized

Hitachi F-4500 spectrofluorometer. The ANS emission was scanned between 400 and 650 nm with an excitation wavelength of 380 nm. The lysozyme adsorption−desorption procedure was performed the same as the CD experiments (concentration of lysozyme at 75 μg/mL). All samples were incubated with ANS for 2 h after the protein desorption procedure. The final concentration of ANS in the lysozyme solutions was 210 μM.

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RESULTS AND DISCUSSION Pore-Expanded MSNs. In recent literature on immobilization of enzyme in mesoporous silica, it is known that matching pore size23,27 and proper surface functionalization28,29 and surface chemistry are crucial for gaining thermal stability and enhanced activity.30 Such fine control of the structure and surface functionality of MSN should be attained first for our purpose. In our previous report, the pore size of MSNs (synthesized with CTAB as the surfactant template) could be efficiently expanded using decane and ethanol as cosolvents.21 Ethanol plays an important role to introduce decane into the CTAB micelles. By controlling the amount of decane and ethanol, we could synthesize a series of MSNs with different pore sizes between 2.5 and 6.0 nm. Here, we show that the morphologies, mesostructures, and suspendibility of the assynthesized MSNs can be controlled and improved by hydrothermal treatment at 80 °C for 1 day (Supporting Information, Figure S1). The gentle hydrothermal process improved the mesostructures. Figure 1 shows the SEM and

Figure 1. (a) SEM image, (b) low-magnification TEM image, and (c, d) HR-TEM images of a pore-expanded MSN functionalized with APTS (MSN-N-4.5).

TEM images of a representative pore-expanded MSN functionalized with APTS (MSN-N-4.5). In the sample notation MSNN-X, N indicates the amine functionalization and X denotes the BJH pore size in nm. These pore-expanded MSNs are disklike in shape, and one can see the well-aligned cylindrical nanochannels with open pore entrances (Figure 1c,d). Meanwhile, the particle size of the MSN could be controlled 6736

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Table 1. Physical Parameters of APTS-Functionalized MSNs sample

d100 (nm)a

a0 (nm)b

DBJH (nm)c

SBET (m2/g)d

Vspt (cm3/g)e

Vmeso (cm3/g)f

PTEM (nm)g

PDLS (nm)h

MSN-N-2.5 MSN-N-4.5 MSN-N-5.6

4.08 5.90 6.49

4.71 6.82 7.50

2.5 4.5 5.6

961 977 1027

1.49 2.13 2.27

0.91 1.28 1.53

79 ± 9 78 ± 10 72 ± 12

117 ± 9 114 ± 10 112 ± 9

a d spacing of the (100) plane, d100 = λ/2 sin(θ100). bValue of the unit cell parameter a0 = 2d100/√3. cPore size calculated by the BJH method from adsorption branch of isotherms. dSurface area calculated by the BET method at relative pressure of P/P0 = 0.05−0.23. eSingle point total pore volume calculated by the nitrogen amount adsorbed at P/P0 = 0.993. fMesopore volume deduced from the BJH adsorption cumulative volume of pores between 1.0 and 15 nm. gParticle size determined from TEM images. hHydrodynamic diameter determined by DLS.

Figure 2. (A) Nitrogen adsorption−desorption isotherms (inset: corresponding pore size distribution plots) and (B) powder X-ray diffraction patterns of (a) MSN-N-2.5, (b) MSN-N-4.5, and (c) MSN-N-5.6.

Figure 3. Zeta potential titration curves of (a) 0.2 mg/mL of lysozyme solution, (b) MSN-N-5.6, (c) MSN-B-5.6, (d) MSN-N-2.5, (e) MSN-N-4.5, and (f) SSN-N.

MSN-Ns were still adsorbed through electrostatic interaction or hydrogen bonding between peptides and MSNs’ silanol groups (Si−OH) (Scheme 1). It is noted that the postfunctionalization method of APTS did not block the pore entrances of MSNs and the isoelectric point of MSN-Ns can be tuned depending on the amount of APTS (Supporting Information, Figure S2). At low temperature (25 °C), APTS could not penetrate into the nanochannels of MSNs and thus only grafted on the external surfaces, confirming the adsorption and desorption strategy (Scheme 1). In addition, for comparison, a nonporous Stöber silica nanoparticle was synthesized and functionalized with APTS (SSN-N) to study the effect of surface geometry on adsorbed lysozyme (Figures 3f and S3, Supporting Information).

Scheme 1. Diagram of Designed Strategy for Lysozyme Adsorption and Desorption from APTS-Functionalized MSNs

silica external surfaces through a strong electrostatic repulsion force. Meanwhile, lysozymes inside or on the entrances of 6737

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Figure 4. Lysozyme adsorption curves on (a) MSN-N-2.5, (b) MSN-N-4.5, (c) MSN-N-5.6, and (d) SSN-N in (A) 20 mM of sodium phosphate buffer at pH 7.4 for 4 h and (B) after desorption in 20 mM of sodium acetate buffer at pH 3.2 for 4 h.

Figure 5. (A) Far-UV CD spectra of lysozyme samples and (B) thermal stability studies of lysozyme secondary structures monitored at 222 nm as a function of temperature: (a) free lysozyme solution and lysozyme adsorbed on (b) MSN-N-2.5, (c) MSN-N-4.5, (d) MSN-N-5.6, and (e) SSN-N. All spectra intensities were normalized to mean residue ellipticity, [θ]MRE.

Lysozyme Adsorption Isotherm. After confirming the appropriate pH ranges for lysozyme adsorption (above pH 6.0) and desorption (below pH 6.0) on MSN-Ns, lysozyme adsorption were first performed at pH 7.4 (Figure 4A). Due to the strong electrostatic interactions between negatively charged MSN-Ns and positively charged lysozyme, Langmuirlike adsorption isotherms with a maximum amount of lysozyme more than 450 μg/mg were obtained. The maximum adsorption of lysozyme on MSN-N depended on the pore size and pore volume. At lysozyme equilibrium concentration of 600 μg/mL, the adsorption amounts of lysozyme on MSN-N4.5 (718 ± 10 μg/mg) and MSN-N-5.6 (830 ± 11 μg/mg) were 161% and 186% higher than that of MSN-N-2.5 (446 ± 17 μg/mg), respectively. The dimension of lysozyme made it difficult to diffuse into the nanochannels of MSN-N-2.5. However, the BJH method is known to underestimate the pore size of mesoporous silica especially for that with small pores.19 Hence, the maximum adsorption of ∼446 μg/mg for MSN-N2.5 is probably from both multilayer adsorption33 on the external surfaces and some lysozyme penetrating into the narrow pores, given its flexibility. On the other hand, the pore sizes and mesopore volumes of MSN-N-4.5 and MSN-N-5.6 were large enough for more lysozyme adsorption. The large adsorption amounts of lysozyme on MSN-N were comparable

to the reported values for lysozyme on SBA-15 with larger pore sizes.34,35 On the other hand, for the SSN-N sample, the maximum adsorption amount of lysozyme was only 195 ± 18 μg/mg due to the absence of mesopores. After the initial adsorption, these silica−lysozyme nanocomposites were transferred to a pH 3.2 buffer for desorption of the adsorbed lysozyme on the external surface of MSN-N samples. The resulting adsorption isotherms are plotted in Figure 4B. One can see the large differences of the lysozyme adsorption curves before and after the external-desorption procedure (Figure 4B). The maximum amounts of adsorption of lysozyme increase with increasing pore size. The values are not much lower than those predicted from the geometry porefilling model proposed by Coppens and co-workers.27 This indicates that the internal mesopores are nearly fully loaded, probably due to the very short channel length in the MSNs.36 The percentages of the remaining lysozyme on silica materials adsorption after the desorption step were quite different between low and high equilibrium concentrations of lysozyme. At a high lysozyme concentration of 600 μg/mL, after desorption, 42.0%, 30.5%, 29.2%, and 57.0% of lysozyme remained on MSN-N-2.5, MSN-N-4.5, MSN-N-5.6, and SSNN, respectively. At a lysozyme concentration of 75 μg/mL, instead, 78.2%, 72.6%, 43.9%, and 62.6.0% of lysozyme 6738

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Table 2. Calculated Secondary Structures of Lysozyme Samples Derived from Far-UV CD Spectra by the CDSSTR Method secondary structure content (%) sample free_LYZ MSN-N-2.5_LYZ MSN-N-4.5_LYZ MSN-N-5.6_LYZ SSN-N_LYZ

α-helix 32.8 20.0 27.3 34.3 15.7

± ± ± ± ±

1.5 1.0 4.9 1.2 0.6

β-sheet 18.0 25.3 19.3 19.0 24.3

± ± ± ± ±

1.1 0.6 3.1 1.0 1.5

β-turn 19.8 27.3 25.0 18.7 25.7

± ± ± ± ±

1.2 0.6 5.3 0.6 0.6

unordered 28.7 27.3 28.7 28.0 34.7

± ± ± ± ±

0.8 1.2 3.1 0.3 0.6

of α-helix were highly correlated to the CD spectra at 222 nm. Lysozyme adsorbed on MSN-N-4.5 retained most of its α-helix in comparison to free lysozyme. In the MSN-N-5.6 sample, the content of α-helix was quite close to that of free lysozyme. In fact, the secondary structure contents (α-helix, β-sheet, β-turn, and unordered) of lysozyme in MSN-N-5.6 were almost the same as that of the free enzyme, as shown in Table 2. By contrast, relatively larger changes in secondary structures were found in the MSN-N-2.5 sample, where some of α-helix was lost during adsorption and more β-sheets were found. The structural integrity of MSN-N-4.5_LYZ is in between the above two samples. In the SSN-N sample, great conformational changes of lysozyme were observed where a large fraction of αhelix was converted to disordered random coil. The study indicated that the integrity of secondary structures was to some degree maintained for the proteins encapsulated in the nanochannels of MSN-N and they are related to pore sizes. The pore size of MSN-N-5.6 probably encapsulates a lysozyme molecule without disturbing its secondary structure, while MSN-N-4.5 squeezed lysozyme a little and led to some structural changes. On MSN-N-2.5 materials, probably most of the lysozymes are on the external or mouths of nanochannels and destabilized more. In terms of the ability to maintain the structural order of the enzymes, the hosts are in the order of MSN-N-5.6 > MSN-N-4.5 > MSN-N-2.5 > SSN-N. Second, we studied the thermal stability of these lysozyme samples in buffered solutions (Figure 5B). The contents of the secondary structures were monitored at 222 nm as a function of temperature. The noisy shape of the curves in Figure 5B probably originated from the light scattering from the MSNs. Nonetheless, the trends of the change of helix structures are discernible. In aqueous solution, free lysozyme as a control sample showed a helix−coil transition temperature at around 76 °C (Figure 5B-a). However, we did not observe an apparent transition of thermal unfolding in lysozymes adsorbed on MSN-N in the temperature range we measured (Figure 5B). Although the secondary structures of lysozyme exhibited various degrees of perturbations after the adsorption− desorption processes on various pore sizes of MSN-N (Table 2), the residual α-helices of lysozyme were mostly preserved in the heating process up to 100 °C. On the other hand, for lysozyme adsorbed on SSN-N, a cooperative transition of unfolding occurred at around 65 °C (Figure 5B-e), less stable than free enzyme. The results of the stability study are consistent with that of the far-UV CD study, i.e., lysozyme adsorbed on the convex surface of SSN-N was more easily denatured (in the α-helical domain). It has been demonstrated that a concave (negative surface curvature) surface could provide an environment that allows better preservation of the native structure of the adsorbed proteins.23 On the basis of the CD data and thermal stability study, we can have some conclusions about lysozyme adsorbed on the MSN-N and SSN-N. For the MSN-N-2.5 sample, because the

remained on MSN-N-2.5, MSN-N-4.5, MSN-N-5.6, and SSNN, respectively. These results should be attributed to the strength of interaction between lysozyme and silica. At high concentration of lysozyme, a large amount of lysozyme was weakly adsorbed through electrostatic forces, which could be easily desorbed when the pH value was lowered. Previously, by exploiting the pH dependence of charges on lysozyme, a pHresponsive valve for controlled drug release from MSN by desorbing encapsulated lysozyme has been developed by Xue et al.19 The trend of larger pore giving higher adsorption is reasonable for the samples. At low concentration of lysozyme, lysozymes were strongly adsorbed on MSN-N and SSN-N, preventing lysozyme to be easily desorbed from silica surfaces. At low concentration, the MSN-N-5.6 seems to give exceptionally low adsorption (at 75 μg/mL). Nevertheless, the adsorption and desorption procedure developed here clearly demonstrated that we can exclude the lysozyme outside mesopores, and thus, the conformation of lysozyme adsorbed inside MSN-N samples could be studied alone. Far-UV CD Spectra and Thermal Stability. Although the loading amount of lysozyme (after desorption) in MSN-N and SSN-N could achieve above 200 μg/mg at high concentration of lysozyme, the lysozyme−material nanocomposites could not be easily suspended in pH 7.4 buffer. In the samples with high loading of lysozyme, because of the charge neutralization, lysozyme-loaded MSNs are easy to aggregate and precipitate. Hence, spectroscopy studies were done at a lower equilibrium concentration of lysozyme (75 μg/mL), i.e., low loading of lysozyme in MSN-N and SSN-N where solutions appeared transparent. In addition, in this concentration, all of the lysozyme was adsorbed on silica materials and no free lysozyme was detectable in the supernatants. Thus, the secondary structures of lysozyme adsorbed on the MSN-N and SSN-N samples can be studied by CD spectroscopy. First, we studied the far-UV CD spectra of the lysozyme samples to examine the secondary structures after adsorption (see Figure 5A). Free lysozyme in buffer solution was used as a control sample. The peak at 222 nm is mainly due to the helical structures of lysozyme, and the one at 209 nm is contributed to the mixed components of α-helices and β-sheets. The α-helix contents (222 nm) of lysozyme adsorbed on MSN-N-2.5 showed a partial denatured profile, and lysozyme adsorbed on MSN-N-4.5 was almost the same as free lysozyme with slightly unfolded structures. In addition, lysozyme adsorbed on MSNN-5.6 showed a similar profile to that of free lysozyme with a more negative mean residue ellipticity. However, for the SSN-N sample, the secondary structure of lysozyme was highly denatured, which was also reported previously by Vertegel et al.8 From these CD spectra, relative contents of various secondary structure contributions in the lysozyme samples were calculated by the CDSSTR method (Table 2). The reconstructed fitting curves were well-fitted to the experimental CD spectra (Supporting Information, Figure S4). The contents 6739

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Figure 6. (A) Relative activities of lysozyme samples before (black) and after (red) hydrothermal treatment at 70 °C for 1 h (residual activities in percentage after hydrothermal treatment were presented). (B) Recyclability of lysozyme adsorbed on MSN-N-5.6.

of the adsorbed protein inside the pores strongly correlates with the curvature and thus influences the catalytic activities. From Figure 5A and Table 2, the secondary structure of lysozyme adsorbed on MSN-N-5.6 was quite similar to the native lysozyme and thus resulted in the highest activity. For MSN-N4.5 and MSN-N-2.5, the activities were lower than that of MSN-N-5.6, probably due to the partially unfolded secondary structures. For the SSN-N sample, the activity of lysozyme was the lowest due to the great loss of secondary structure after adsorption. Strong interaction between lysozyme and flat silica surfaces could significantly affect the structure and function of lysozyme.8 Furthermore, after hydrothermal treatment at 70 °C for 1 h, the MSN-N-5.6 and MSN-N-4.5 samples retained more of their activities, which are well-correlated to the thermal stability studies shown in Figure 5B-c,d. Lysozymes confined in the nanochannels are prevented from denaturation and thus maintained their functions. However, lysozyme adsorbed on MSN-N-2.5 showed low residual activity (35%), similar to that of free lysozyme, which confirmed that most of the enzymes was located outside the pores of MSN-N-2.5. Thus, when lysozyme adsorbed on MSN-N-2.5, the enzyme molecule could not very easily diffuse into the small channels and thus could not be well-protected. It has been reported that lysozyme was most likely to adsorb on silica surfaces, landing with the most positively charged patch, which is at the opposite side of the active site.38,39 Probably, because the active site is open toward solution, the lysozyme in MSN-N-2.5 shows higher activity (before hydrothermal treatment) compared to lysozyme confined in MSN-N-4.5. For the SSN-N sample, after hydrothermal treatment, lysozyme showed very low residual activity, which was reasonable due to its unfolding transition near 70 °C found by CD spectroscopy (Figure 5B-e). Compared to the size of lysozyme molecules, SSN-N (∼60 nm) is very large in diameter. Hence, SSN-N appears like a flat surface for lysozyme adsorption. In this situation, the contact surface area between lysozyme and SSN-N tends to spread due to strong electrostatic interactions, thus resulting in the unfolded structures of lysozyme.8 Then, we examine the reuse of the catalyst [email protected], since this sample showed the best catalytic activity and thermal stability. The catalytic activity remained ∼70% of its

lysozyme molecule is too large to fully diffuse into its nanochannels, most of the lysozyme would be probably at the openings of mesopores. Under this situation, more surface contact between lysozyme and silica walls was responsible for the partial denaturation of lysozyme. Nevertheless, lysozyme showed no further unfolding in the heating process from 20 to 100 °C, indicating that secondary structures were protected to some extent. It is assumed that a part of flexible structure of lysozyme may diffuse into the mesopores, even with a small pore size of MSN-N-2.5. For the MSN-N-4.5 samples, the dimensions of lysozyme molecules are matching most with the pore diameter of MSN-N-4.5, and thus, lysozyme not only retained high content of secondary structure in adsorption but also showed great thermal stability. For MSN-N-5.6, lysozyme was supposed to be more easily adsorbed into the nanospaces of MSNs. It was reported that when lysozyme was dissolved in glycerol, the secondary structures were enhanced due to hydrogen bonding with the environment.37 The silanol groups inside the nanochannels probably formed hydrogen bonds with lysozyme, resulting in the enhanced secondary structure on farUV CD spectra (Figure 5A-d). Lysozyme confined in MSN-N5.6 showed significantly enhanced thermal stability up to 100 °C (Figure 5B-d). These results indicated that lysozyme could diffuse into the nanochannels of MSN-N-5.6, and the unfolding of lysozyme was limited. In comparison, the lysozyme adsorbed on SSN-N sample showed both denatured secondary structures and poor thermal stability; we thus infer that lysozyme adsorbed on porous silica surfaces was more stable. Activity Assays of Confined Lysozyme. Lysozyme activity assay was performed with the trisaccharide 4-MU-β(GlcNAc)3 as substrate. A representative fluorescence spectra of catalyzed product (coumarin) as a function of reaction time (0−80 min) is shown in Figure S5 (Supporting Information). The product’s fluorescence intensity against time was plotted and the reaction rate of each sample was determined from the slope of the linear plot (Supporting Information, Figure S5). Relative activities of free lysozyme and adsorbed lysozyme before and after hydrothermal treatment at 70 °C for 1 h are shown in Figure 6A. It is found that lysozyme adsorbed on all MSN-N showed higher activities than free lysozyme. This has been termed as enzyme superactivity in the literature.9−11 Sang et al. previously reported superactivity results when lysozyme was adsorbed in SBA-15.23 They found that the conformation 6740

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Figure 7. FL spectra of ANS binding experiments. (A) ANS blank solutions: (i) 210 μM of ANS in pH 7.4 buffer solution, (ii) 210 μM of ANS in MSN-N-2.5 solution, (iii) 210 μM of ANS in MSN-N-4.5 solution, and (iv) 210 μM of ANS in MSN-N-5.6 solution. (B) ANS−LYZ solutions (ANS was 210 μM): (a) free LYZ, (b) MSN-N-2.5_LYZ, (c) MSN-N-4.5_LYZ, and (d) MSN-N-5.6_LYZ.

original after five cycles (Figure 6B). The decrease in activity in cycle 4 may be due to some loss of enzyme after extensive washing. Anyway, we can conclude when lysozyme are wellprotected inside the pores of MSN-N-5.6, the catalyst can be recycled well. Finally, we would like to explore the cause of the superactivity of mesopore-confined lysozyme, especially in the case of [email protected]. In general, supported enzyme showed lower activity than free enzyme in solution. For example, enzymatic activities of lysozyme adsorbed on silica nanoparticles were found to be less than that of free enzyme. The fraction of activity loss correlated well with the decrease in α-helix content upon adsorption on silica surface.8 However, when confined inside properly chosen nanopores, superactivity has been found in enzymes adsorbed in mesoporous silica.11,23 The superactivity needs to be explained because subactivity instead of superactivity would have been expected at least for the reason of transport barrier in-and-out of the pores if not denatured. In comparing lysozyme in MSN-N-5.6 versus free enzyme, we have shown that the secondary structure of the poreconfined enzyme is almost the same, while it showed about 2.75 times higher activity. Here we employed a fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS), to study the protein binding site. ANS has been extensively used in probing the tertiary structure of molten globules of protein, where internal hydrophobic surface of protein becomes exposed to ANS upon its formation.40,41 The observed fluorescence changes of ANS, a blue shift of fluorescence emission maxima and the increase of fluorescence intensity, are generally attributed to the hydrophobicity of a binding site. Figure 7 shows the results of ANS binding experiments. Figure 7A gives the fluorescence spectra of blank solution without lysozyme. No spectral shift was detected when ANS interacted with the silica surface of MSN-N compared to ANS in buffer solution. When lysozyme is adsorbed onto MSN-N, they show a blue shift of ∼15 nm wavelength and a small increase of intensity compared to free lysozyme (Figure 7B). These spectral changes indicate a minor change of tertiary structure of lysozyme such that ANS was exposed more to the interior hydrophobic surface. It should be noted that the change of the fluorescence of ANS is much smaller than those normally observed in a transition to a molten globule protein. We actually do not

expect that kind of high degree of change of tertiary structure; if so, the enzyme would have been deactivated. In our case, probably the pocket to the active site opened just enough to gain more access while the catalytic site is still active. We should note that there are a number of conventional explanations for the superactivity in confined enzyme.42 Here, we would like to examine two alternative explanations and our reasons for rejecting them: (a) aggregation of the ‘‘free” lysozyme [It could be that the “soluble” free lysozymes consist of multimeric aggregations of enzymes and we were only comparing between the “immobilized” monomeric enzyme and multimeric enzyme in solution. The multimeric enzyme gave lower activity. The blue shift of ANS in confined mesopores reflects the more exposed hydrophobic surface in confined monomeric lysozyme than in “free” lysozyme. For this possibility, we performed size exclusion chromatography analysis of the soluble lysozyme in the same pH buffer (7.4) solution condition as in catalysis study. The result (Supporting Information, Figure S7) shows that our “free” enzyme consists of only monomeric lysozyme. Neither dimer nor any higherorder forms of lysozyme were detected.] and (b) effect of substrate or product partition [An enhanced catalytic activity immobilization of enzymes on a porous support may be due to partition of the reactant (toward the enzyme environment) and product (away from the enzyme environment). In our case, the substrate 4-MU-β-(GlcNAc)3 contains two groups, the hydrophilic triacetylchitotrioside and the hydrophobic methylumbelliferyl group connected by a −O− linkage. If 4-MU-β(GlcNAc)3 were concentrated inside the mesopore, then the product GlcNAc, after breaking of the O-linkage, should bind to silica even more strongly. One would have an inhibition of the reaction due to product saturation of the pore, instead of a promotion. Apparently, this did not occur.]. Therefore, we believe a structural explanation (tertiary structure based on ANS fluorescence) of the superactivity in confined lysozyme is more likely. Further spectroscopic studies, such as NMR, on pore-confined enzyme would be highly desirable to gain deeper understanding of the structure−activity relationship in the confined enzyme. At present, knowledge about the microenvironment of crowded enzyme is still rather scant. One needs to learn more about the state of enzyme hydration and conformation in a crowded environment in order to really understand its structure−activity relationship. This study has revealed many hidden elements of protein 6741

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The Journal of Physical Chemistry C immobilization and provided us with a way of understanding of the interactions between MSNs and proteins. In this work, we studied the effect of pore size design for optimum catalytic action of the encapsulated enzyme. Our studies clearly demonstrate that we are able to enhance the stability and catalytic activity of lysozyme by immobilizing on matching porous surface of nanochannels of MSNs. Previously, we reported that cytochrome c embedded in the channels of micrometer-sized mesoporous silica could be protected against unfolding and loss of activity when pore size matched.43,44 This work again shows the importance of pore size matching. Further developments would be possible in designing the microenvironments of the nanopores, for example, by using periodic mesoporous organosilica (PMO)45,46 or by surface modification with other functionalities.47

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REFERENCES

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CONCLUSIONS This work shows that the enzyme immobilization can improve not only the enzyme stability but also the enzymatic activity by protecting it against thermal denaturation and subtle structural changes. We found that the stability and catalytic activity critically depend on the pore size and the surface curvature, most favorably on the concave surface of MSNs, especially when lysozyme is confined in MSN-N-5.6 with pore size close to its own dimensions, and thus, the native conformation is stabilized. Fluorescence spectra of adsorbed ANS probe indicate increased access of the hydrophobic interior of the lysozyme in the MSN sample with 5.6 nm pore size. We further found superactivity in our enzyme−nanoparticle when the pore size of MSNs is comparable to the dimension of the protein. The superactivity was attributed to the crowded microenvironment in the nanopores of mesoporous silica. Our studies show that the concave curvature of MSNs promotes the interaction between protein and nanochannels surfaces and endow the crowding of proteins on the surface of high porosity comparable to free enzymes in cytosol. The CD studies of the composite nanomaterials allowed us to examine the stability and the secondary structure of lysozyme. We found that the α-helical domain was sensitive to the protein denaturation in the cases of small nanochannels, i.e., less than the dimension of lysozyme. Most importantly, the confinement effect on lysozyme, specifically in MSN-N-5.6 nm samples, limited the unfolding and showed significantly enhanced thermal stability up to 100 °C. Thus, our studies indicated that the enhanced catalytic activity was mainly arising from the crowding effect. ASSOCIATED CONTENT

S Supporting Information *

Material sources and characterization; additional information of CD spectroscopy setting; size-exclusion chromatography; TEM images, XRD patterns, DLS measurments, and zeta-potential titration curves of control silica samples. This information is available free of charge via the Internet at http://pubs.acs.org

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ACKNOWLEDGMENTS

This work was supported by the National Science Council of Taiwan. We thank Yun Mou and Yu-Chieh Huang for performing the size exclusion chromatography experiment.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 6742

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