Immobilization of Lysozyme Onto Pore-Engineered Mesoporous Alsba-15

Immobilization of Lysozyme onto Pore-Engineered Mesoporous AlSBA-15

Masahiko Miyahara, Ajayan Vinu,* and Katsuhiko Ariga*

Affiliation:

World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS)

Mailing Address:World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan.

Phone: +81-29-860-4597

Fax: +81-29-860-4832

E-mail:

E-mail:

Abstract

In this research, hydrothermally stable mesoporous AlSBA-15 materials have been used as adsorbents for systematic research on the lysozyme adsorption. Stability of the AlSBA-15 adsorbents and the lysozyme molecules after the adsorption experiments for several days in aqueous solutions were confirmed by X-ray diffraction (XRD) measurement and FT-IR spectroscopy, respectively. The amount of the lysozyme adsorption can easily be controlled by the pore diameter and pore volume the mesoporous adsorbent, but an unreasonable effect of the surface area on the protein adsorption capacity was observed. The results of the effect of the pore diameter on the lysozyme adsorption suggest that the adsorption might partially be influenced by kinetically favorable edge-on type orientation on the confined mesopore. However, the final adsorption amount of the lysozyme can be well regenerated by models based on the side-on adsorption in dense packing. The present research also confirms the importance of appropriate "pore-engineering" for immobilization of bio-function on mesoporous materials.

Keyword:mesoporous silica, protein, lysozyme, AlSBA-15, pore volume, pore diameter.

1. INTRODUCTION

Material conversion in biological systems is performed by enzymes with surprisingly high efficiency and specificity under very mild condition. Mimicking high functions of the enzymes can be regarded as one of the ultimate goal for currently developing catalysts. Some of supramolecular compounds, which are often called artificial enzymes,1-5regenerate parts of enzyme functions, but their performances are not always satisfactory especially in practical usages. Instead, hybridization of naturally occurring enzymes with artificial membranes such as lipid bilayers,6,7 Langmuir-Blodgett (LB) films,8,9 and layer-by-layer (LBL) assemblies10-17 provides molecularly engineered devices such as sensors, reactors, and signal converter. However, most of them are not tolerant to harsh conditions due to fragile nature of support structures. Therefore, immobilization of enzymes onto rigid inorganic frameworks ishighly necessary for many practical applications.

Mesoporous silica,18-24with highly regular pore geometries, has high potential as a support for enzyme immobilization and its practical applications based on their mechanical stability as well as huge surface area, and high reliability of pore dimensions are highly expected. Several attempts to immobilize biomaterials such as amino acids,25-27 vitamins,28 peptides,29 and proteins30-47 to mesoporous silica materials have been investigated. However, silica structures tend to be, more or less, degraded through hydrolysis of siloxane linkages, resulting in inappropriateness in long use in water and/or unreliability of data collected in aqueous media. Very recently, it has been demonstrated that mesoporous carbon materials have capability to accommodate proteins in their pore,48,49 but these researches are now in pioneering stage.

In this research, we have used AlSBA-15 mesoporous silica materials as adsorbents for lysozyme adsorption. Incorporation of aluminum atom in mesoporous silica structure drastically increased its hydrothermal stability due to formation of Si-O-Al bonds,50 which are more resistant to attack from water as compared with Si-O-Si bond. In addition, content of micropore in AlSBA-15 materials is far less as compared to that of SBA-15, which provide more reliable analyses for effect of pore geometry on lysozyme adsorption. Furthermore, charge density can be well controlled in AlSBA-15 materials through introduction of negative charges on the surface by incorporating aluminum atom in tetrahedral positions. Recently, Vinu et al. have reported the direct synthesis of AlSBA-15 mesoporous silica materials and found that the pore diameter of the above materials can be easily tuned by simply changing the synthetic temperature.51 Based on these advantages of AlSBA-15 mesoporous silica in protein adsorption, we have systematically investigated adsorption of lysozyme onto "pore-engineered" AlSBA-15 and proposed models of pore-filling by lysozyme molecules.

2. EXPERIMENTAL SECTION

2.1. Materials and Syntheses

Tetraethyl orthosilicate, sucrose and tri-block copolymer poly (ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic P123, molecular weight = 5800, EO20PO70EO20)were obtained from Aldrich. Inorganic salts for buffer preparation were purchased from Wako Pure Chem. Chicken egg white lysozyme (E.C. 3.2.1.17) was obtained from Sigma and used without further purification.

Details of AlSBA-15 syntheses were described previously.51 A set of AlSBA-15samples with nSi/nAlatomic ratio of 45was prepared by varying thesynthesis temperature from 110 to 130 °C. The obtained samples werelabeled AlSBA-15-X, where Xdenotes the synthesis temperature (X = 110, 120, and 130). Detailed characterization of these AlSBA-15 materials can be found in the previous paper. The textural parameters of the AlSBA-15 materials are briefly summarized in Table 1.

2.2. Characterization

The powder X-ray diffraction (XRD)patterns of mesoporous materials were collected on aRigaku diffractometer with use of Cu K( = 0.154 nm)radiation. The diffractograms were recorded with a 2step size of 0.01and a step time of 10s. Nitrogen adsorption and desorption isotherms were measuredat -196 C on a Quantachrome Autosorb 1 sorption analyzer. All samples before protein adsorption were outgassed at 250C for 3 h prior to the nitrogen adsorption measurements, whilethe protein adsorbed samples were outgassed at 40 C for 24 h. The specific surface area was calculated with use of theBrunauer-Emmett-Teller (BET) method. The pore size distributionswere obtained from the adsorption and desorptionbranch of the nitrogen isotherms by both the Barrett-Joyner-Halenda and the Nonlocal Density Functional Theory (NLDFT)methods. The mercury porosimetry data were collected on aMicromeritics Autopore II 9220 instrument. FT-IR spectra of lysozyme before and after theprotein adsorption were recorded on a Nicolet Nexus 670instrument by averaging 200 scans with a resolution of 2 cm-1measuring in transmission mode by using the KBr self-supportedpellet technique. The spectrometer chamber was continuouslypurged with dry air to remove water vapor.

2.3. Adsorption Study

Method for adsorption study of lysozyme onto AlSBA-15 mesoporous silica materials are schematically illustrated in Figure 1. A series of standard lysozyme aqueous solutionswasprepared by dissolving different amounts of lysozyme in 25 mM buffersolutions: potassium phosphate buffer for pH 6.5 condition; sodium bicarbonate buffer for pH 9.6 and pH 11.0 conditions. In each adsorption experiment, 20 mg of the different AlSBA-15 adsorbents was suspended in 4g of the respective lysozyme solution. The resulting mixture was continuously shaken ina shaking bath with a speed of 160 shakes/min at 20ºC untilequilibrium was reached (typically 96 h). The amount of lysozyme adsorbed was calculated by subtracting the amount found inthe supernatant liquid after adsorption from the amount of lysozyme present before addition of the adsorbent by UV absorption at281.5 nm. Calibration experiments were done separately beforeeach set of measurements with the lysozyme solutions of differentconcentrations buffered at the same pH as the isotherm. Centrifugation prior to the analysis was used to avoid potentialinterference from suspended scattering particles in the UV-Visanalysis.

3. RESULTS AND DISCUSSION

3.1. Basic Behaviors of Lysozyme Adsorption onto AlSBA-15

Since lysozyme tends to denaturate at high pH conditions, the adsorption study was carried out in pH range between 6.5 and 11.0. Adsorption profiles of lysozyme onto various AlSBA-15 mesoporous silica materials are summarized in Figure 2. All the isotherms obey Langmuir-type adsorption (see eq. (1)), indicating that the inner surface of AlSBA-15 pores are covered with the monolayer of the lysozyme molecules.

ns = K nm [lysozyme] / (1 + K [lysozyme])(1)

In this equation, ns, nm, and K represent the amount of adsorbed lysozyme, monolayer adsorption capacity, and binding constant, among which we mainly discuss the monolayer adsorption capacity (see Table 2). Effect of solution pH on the monolayer adsorption capacity is summarized in Figure 4. For all the AlSBA-15 adsorbents, the maximum adsorption was observed at around pH 11.0, which is very closed to the isoelectric point of lysozyme.52 Near the isoelectric point,the net charge of the protein is low and the Coulombic repulsiveforce between the protein molecules is minimal, where a closer packing of the protein molecules would lead to themonolayer capacity increases. The adsorption maximum at the isoelectric point of lysozyme can also be explained bythe change of the limiting area per molecule for lysozyme with solutionpH. It has been reported that the area per molecule of lysozyme insolution having a pH near the isoelectric point is similar to thearea per molecule of lysozyme in its crystallized state (13.5 nm2),whereas it expands to 26.6 nm2 at a solution pH of 4.52 This change of the lysozyme size is consistent with the most compact adsorption at the isoelectric point.

Structure of AlSBA-15 adsorbents before and after the protein adsorption study was investigated by XRD measurement (Figure 4). All samples exhibit the XRD pattern typically observed for AlSBA-15, consisting of a strong (100) reflection at a low angleand two small peaks at higher angles. The positions of these peaks were not fundamentally shifted even after the adsorbents were soaked in basic aqueous solution at pH 11 for 4 days. This confirms the retention of the hexagonal mesoporousstructure of AlSBA-15 even after the adsorption experiments. Interestingly, the intensity of the peaks decreased as compared to theparent AlSBA-15 materials upon the lysozyme adsorption. This may notbe interpreted as a severe loss of structural order, but it is likelythat larger contrast in density between the silica walls and theopen pores relative to that between the silica walls and the lysozyme molecule inside the pores is responsible for the observeddecrease in intensity. Therefore, reduction in the intensity of XRD peaks after protein adsorptionis mainly due to the tight packing of lysozyme molecules inside themesopores of aluminosilicateadsorbents.

Structure of lysozyme, before and after the adsorption experiment, was similarly investigated using FT-IR spectroscopy. The spectra at the region of amide I and amide II bands, which are sensitive to protein denaturation, are shown in Figure 5. The peak positions of the amide I and amide II of lysozyme loaded on AlSBA-15 mesopore are not basically shifted as compared with those observed for pure lysozyme. Intensity ratios of these peaks (amide I / amide II) are 1.3, 1.3, and 1.2 for lysozyme on AlSBA-15-110, AlSBA-15-120, and AlSBA-15-130, respectively, while 1.2 was observed for pure lysozyme. Absence of any changes in these peaks among the tested samples indicates that adsorption onto the AlSBA-15 adsorbents does not cause serious changes of the lysozyme secondary structures. The adsorbed lysozyme molecules are quite stable and did not denaturate upon adsorption onto AlSBA-15 mesoporous silica materials.

3.2. Effect of Pore Geometries on Lysozyme Adsorption

Asthe lysozyme dimension at its isoelectric point is rather close to that crystallographically obtained,52 adsorption data obtained at pH 11.0 are mainly used for detailed discussion in later section. The monolayer lysozyme adsorption capacities of three different AlSBA-15 adsorbents are plotted as functions of various structural parameters (Figure 6). It is important to note that the monolayer adsorption capacity decreased as the surface area increased (Figure 6A). The surface area would not be a decisive factor for protein adsorption onto mesoporous structure, because the total surface area always includes unavoidable contribution from micropores, which cannot accommodate large lysozyme.

The monolayer adsorption capacity increased as pore volume increased (Figure 6B), suggesting that AlSBA-15 material with larger pore volume has higher capacity for the lysozyme accommodation. It sounds reasonable but simple, because their relation is not linear. The adsorption capacity of lysozyme showed clear linear relation with pore diameters that were obtained from nitrogen adsorption isotherm (Figure 6C) and mercury porosimetry (Figure 6D). The mercuryporosimetry is useful in evaluation of size of pores with diameter more than 3 nm,51 but the both plots have similar tendency. Furthermore, occupation of AlSBA-15 pores by lysozyme was evaluated from the pore volume of AlSBA-15 materials and molecular volume of lysozyme (17.8 nm3).49 The pore occupation by lysozyme is in range between 20 - 30% and showed apparent dependence on pore diameter. Therefore, amount of lysozyme adsorption is not only determined simply by spatial volume of the mesopore and is also influenced by the pore diameter.

Short axis of the lysozyme molecule is ca 3 nm that is sufficiently smaller than the pore diameters of the AlSBA-15 materials (more than 10 nm). The lysozyme molecules would not seriously interfere each other when they diffuse into the AlSBA-15 pores with orientating long axis (ca 4.5 nm) of lysozyme along pore extension direction. However, the lysozyme molecules may experience steric interference if they are inserted into the pore through directing their long axis perpendicular to pore extension axis. Previous researches suggested reorientation behavior of the lysozyme adsorbed on quartz.53 Lysozyme kinetically tends to adsorb on the surface in its edge-on orientation (Figure 8A) in the initial stage, but the orientation changes into a side-on fashion toward equilibrium (Figure 8B). The edge-on adsorption in the mesopore would cause some steric hindrances between adsorbed lysozyme molecules, leading to unavoidable effect of pore size on the lysozyme adsorption. Therefore, the observed effect of the pore diameter would be originated from the initial orientation (edge-on) of the lysozyme adsorption.

Adsorption amountsof lysozyme are calculatedthrough models depicted in Figure 9 where several packing models are proposed both for edge-on and side-on orientation. In these models, AlSBA-15 materials are assumed to have cylindrical pores with a diameter of x nm, and an ellipsoidal shape with dimension of a x b x b nm is assigned to the lysozyme molecule (Figure 9A). For the edge-on orientation on the lysozyme adsorption, the following equations are given as pore occupation (Figure 9B).

4ab / 3x2 for doublet model(2)

8ab /3x2 for qualtet model(3)

4ab / x2 for sextet model(4)

16ab /3x2 for octet model(5)

From the reported data, a and b are assigned to 3 and 4.5 nm, respectively.54 In the case of the AlSBA-15-130 adsorbent, x can be 12.5 nm as estimated from the nitrogen adsorption data. These values lead to occupation degree of AlSBA-15 mesopores by the lysozyme molecules as 0.115, 0.230, 0.346, and 0.461 for the doublet, quartet, sextet, and octet models, respectively. Among them, the octet model are over crowded and may not be possible packing. Even sextet model showed pore occupation value higher than that experimentally obtained (0.312, see Figure 7).

Similar models are also estimated for the side-on adsorption (Figure 9C), with the following equations as pore occupation by the lysozyme molecules.

4b2 / 3x2 for doublet model(6)

8b2 / 3x2 for quartet model(7)

4b2 / x2 for sextet model(8)

16b2 /3x2 for octet model(9)

Applying the same structural parametersto these models results in the pore occupation degree as 0.077, 0.154, 0.230, and 0.307 for the doublet, quartet, sextet, and octet models, respectively. Well packed octet model with the side-on orientation provides the pore occupation value very closed to the experimental value. Therefore, the final adsorption amount of lysozyme in the AlSBA-15 mesopore would be determined by good packing with more stable side-on orientation, even though the adsorption process through the kinetically favorable edge-on orientation may cause certain effect of pore diameter on the adsorption amount. Relation of the lysozyme adsorption amount and its orientation cannot be decisively interpreted by the experimental dada presented here, but theoretically obtained values from these models fall into the reasonable range as compared with the experimental value. This fact confirms validity of the model consideration on protein adsorption in mesopore spaces.

4. CONCLUSION

In the present research, adsorption behaviors of lysozyme onto AlSBA-15 mesoporous silica materials, which is stable for several days even in aqueous medium, have been investigated. The adsorption amount is influenced by pore diameter and pore volume, while the surface area does not seem to be important factor on the protein adsorption. The pore occupation by the lysozyme molecules showed certain dependence on pore diameter, although the pore diameters of the AlSBA-15 adsorbents are sufficiently larger than the short axis of the lysozyme molecule. Kinetically favorable edge-on orientation on the silica inner wall might have some influence on regulation of the adsorbedamount. However, the final amount of the lysozyme adsorption can be well regenerated by models based on the side-on adsorption in dense packing. The present research confirms importance of use of stable adsorbent for the reliable experiments of the protein adsorption onto mesoporous silica, as well as the importance of consideration based on appropriate models for further understanding protein packing and orientation in mesopores. The obtained knowledge must be indispensable for immobilization of bio-function on the “Pore-Engineered” mesoporous materials.

Acknowledgements

A. Vinu is grateful to Prof. Y. Bando and Special Coordination Funds for PromotingScience and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government for the award of ICYS Research Fellowship, Japan. This work is also partially supported by Grant-in Aid for Scientific Research onPriority Areas (No. 17036070 “Chemistry of CoordinationSpace”) from Ministry of Education, Science, Sports, andCulture, Japan.

References

1.R. Breslow, Science218, 532 (1982).

2.K. Ariga, and E. V. Anslyn, J. Org. Chem. 57, 417 (1992).

3.J. Smith, K. Ariga, and E. V. Anslyn, J. Am. Chem. Soc. 115, 362 (1993).

4.D. M. Kneeland, K. Ariga, V. M. Lynch, C. Y. Huang, and E. V. Anslyn, J. Am. Chem. Soc. 115, 10042 (1993)

5.K. Ariga, T. Nakanishi, J. P. Hill, M. Shirai, M. Okuno, T. Abe, and J. Kikuchi, J. Am. Chem. Soc.127, 12074 (2005).

6.J. Kikuchi, K. Ariga, T. Miyazaki, and K. Ikeda, Chem. Lett. 253 (1999).

7.J. Kikuchi, K. Ariga, and K. Ikeda, Chem. Commun. 547 (1999).

8.Y. Okahata, T. Tsuruta, K. Ijiro, and K. Ariga, Langmuir 4, 1373 (1988).

9.Y. Okahata, T. Tsuruta, K. Ijiro, and K. Ariga, Thin Solid Films 180, 65 (1989).

10.Y. Lvov, K. Ariga, and T. Kunitake, Chem. Lett. 2323 (1994).