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Porous materials
Simple Synthesis of Hierarchically Ordered Mesocellular Mesoporous Silica Materials Hosting Crosslinked Enzyme Aggregates Jinwoo Lee, Jungbae Kim,* Jaeyun Kim, Hongfei Jia, Moon Il Kim, Ja Hun Kwak, Sunmi Jin, Alice Dohnalkova, Hyun Gyu Park, Ho Nam Chang, Ping Wang, Jay W. Grate, and Taeghwan Hyeon*
Hierarchically ordered mesocellular mesoporous silica materials (HMMS) were synthesized using a single structure-directing agent. The mesocellular pores are synthesized without adding any pore expander; the pore walls are composed of SBA-15 type mesopores. Small-angle Xray scattering revealed the presence of uniform pore structures with two different sizes. Using HMMS as a nanoscopic template, hierarchically ordered mesocellular mesoporous carbon (HMMC) and polymer (HMMP) materials were synthesized. HMMS was used as a host for enzyme immobilization. To improve the retention of enzymes in HMMS, we adsorbed enzymes, and then employed crosslinking using glutaraldehyde (GA). The resulting crosslinked enzyme aggregates (CLEAs) show an impressive stability with extremely high enzyme loadings. For example, 0.5 g a-chymotrypsin (CT) could be loaded in 1 g of silica with no activity decrease observed with rigorous shaking over one month. In contrast, adsorbed CT without GA treatment resulted in a lower loading, which further decreased due to continuous leaching of adsorbed CT under shaking. The activity of crosslinked CT aggregates in HMMS was 10 times higher than that of the adsorbed CT, which represents a 74fold increase in activity per unit weight of HMMS due to higher CT loading.
[*] Dr. J. Kim, Dr. J. H. Kwak, A. Dohnalkova, Dr. J. W. Grate Pacific Northwest National Laboratory Richland, WA 99352 (USA) Fax: (+ 1) 509-375-5106 E-mail:
[email protected] Dr. J. Lee, J. Kim, S. Jin, Prof. Dr. T. Hyeon National Creative Research Initiative Center for Oxide Nanocrystalline Materials and School of Chemical Engineering Seoul National University, Seoul 151-744 (Korea) Fax: (+ 82) 2-888-1604 E-mail:
[email protected]
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Keywords: · crosslinking · enzymes · mesoporous materials · silica · template synthesis
H. Jia, Prof. Dr. P. Wang Department of Chemical Engineering University of Akron, Akron, Ohio 44325 (USA) M. I. Kim, Prof. H. G. Park, Prof. H. N. Chang Department of Chemical and Biomolecular Engineering Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea) Supporting information for this article is available on the WWW under http://www.small-journal.com or from the author.
DOI: 10.1002/smll.200500035
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1. Introduction Mesoporous materials with pore sizes ranging from 2 to 50 nm have attracted considerable attention because of their many applications involving large molecules, which cannot be accomplished using conventional microporous zeolitic materials.[1] Amphiphilic block copolymers, along with surfactant self-assemblies, have proven to be valuable supramolecular templates for the synthesis of ordered mesostructured materials.[2] Mesocellular siliceous foam (MCF) with ultra-large mesocellular pores connected by mesoporous windows have been fabricated using tri-block copolymers as a structure-directing agent, and 1,3,5-trimethylbenzene (TMB) as a pore expander.[3] Complementary pores, formed by the penetration of ethylene oxide (EO) groups, are present in the walls of the MCF.[4] However, these complementary pores are disordered micropores smaller than 2 nm. Recently, active research has been conducted on the synthesis of hierarchically ordered porous materials.[5] Generally, dual templates were used to synthesize such materials. Polystyrene (PS) beads have frequently been used for the generation of macropores, while smaller-sized templates were used for the mesopores. In a previous study, macroporous silica materials with zeolitic walls were synthesized using PS beads and tetrapropyl ammonium hydroxide (TPAOH) as dual templates.[5a] Preformed silicalite nanoparticles and starch gel were used to make macroporous/microporous materials.[5b] Macro/mesoporous silica has been produced using PS beads and low-molecular-weight surfactants or amphiphilic triblock copolymers as dual templates.[5c,d] Monolithic silica[5e] and silica films[5f] with bimodal pore structures were also prepared. Bimodal super-microporous and macroporous silica material was synthesized using a PS bead packing and an ionic liquid as dual templates.[5h] Trimodal porous silica was prepared using PS beads for macropores, a block copolymer for large mesopores, and an ionic liquid for small mespores, respectively.[5i] Recently, bimodal porous materi-
Editorial Advisory Board Member Taeghwan Hyeon received his BS (1987) and MS (1989) in Chemistry from Seoul National University, Korea. He obtained his PhD from the University of Illinois at Urbana-Champaign (1996). Since he joined the faculty of the School of Chemical and Biological Engineering of Seoul National University in 1997, he has been focused on the synthesis of uniformsized nanocrystals and new nanoporous carbon materials. He is currently Director of the National Creative Research Initiative Center for Oxide Nanocrystalline Materials. Over the past five years, he has published more than 70 papers in prominent international journals. He has received several awards, including the T. S. Piper Award from the University of Illinois, the Korean Young Scientist Award from the Korean President, and the Dupont Scientist Award.
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als were synthesized using a single template. Antonelli reported the synthesis of macro/mesoporous niobium oxide molecular sieves by adding a large amount of NaCl and using a single dodecylamine surfactant.[6] The author claimed that NaCl played a crucial role in the formation of the large-sized vesicles that led to the generation of the macropores. More recently, bimodal porous materials of titania and zirconia with wormhole-like mesopores and a funnellike macrostructure were synthesized using single-surfactant decaoxyethylene cetyl ether (C16(EO)10).[7] The generation of the macropores was attributed to the vesicle-type supermicelles formed by the aggregation of unreacted excess surfactants. Herein, we report on the synthesis of a mesocellular silica foam with ordered mesoporous walls, designated as hierarchical mesocellular mesoporous silica (HMMS), using an amphiphilic triblock copolymer as a single structure-directing agent. The synthetic method for HMMS is very simple and cost-effective. Generally, to make mesocellulartype pores, a pore expander[3] such as trimethylbenzene is required. However, in this synthesis, the mesocellular pores are synthesized without adding an additional pore expander. Interestingly, the walls of the cellular pores in HMMS are composed of SBA-15-type pores. The final pore structure of HMMS is a mixed structure of mesocellular silica foam (MCF)[3] pores and one-dimensional SBA-15 pores.[2a] Using HMMS silica as a template, hierarchical mesocellular mesoporous carbon and polymer materials were successfully fabricated. As a possible application of HMMS, we attempted to immobilize enzymes in HMMS. Mesoporous materials have attracted much attention for enzyme immobilization,[8, 9] and much effort to improve both enzyme loading and stability has been made.[9] In this paper, we have developed crosslinked enzyme aggregates (CLEAs)[10] in HMMS, which employs the adsorption of enzymes followed by enzyme crosslinking using glutaraldehyde. Since the multipoint attachment of enzyme molecules is well-known to stabilize activity by preventing enzyme denaturation,[11] we anticipated that CLEAs in HMMS would stabilize enzyme activity. During the measurement of enzyme stability, all immobilized enzymes were incubated under rigorous shaking. Even though shaking is required in a conventional enzyme reactor using immobilized enzymes, few reports have analyzed the longevity of immobilized enzymes in mesoporous materials under shaking. This unprecedented approach (using shaking) was possible because CLEAs in HMMS clearly stabilized the enzyme activity.
2. Results and Discussion 2.1. Synthesis and Characterization of HMMS The structure of HMMS is presented in Figure 1. Mesocellular siliceous foam (MCF)[3] is composed of large cells 2005 Wiley-VCH Verlag GmbH & Co. KGaA, D-69451 Weinheim
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Figure 1. Cartoon showing that the structure of HMMS is a mixed structure of mesocellular type pores and one-dimensional-channel SBA-15-type pores.
(> 20 nm) and connecting windows. To make MCF, oil-inwater microemulsions were used as structure-directing agents for structure assembly. Hexagonally ordered mesoporous silica[2a] is synthesized using P123 ((EO)20(PO)70(EO)20) as a structure-directing agent. The addition of trimethylbenzene (TMB) to a P123 solution as a pore expander converts hexagonally ordered structures to mesocellular structures, which exhibit spherical pores.[3b] HMMS has two interesting pore structures in one mesostructured primary particle. In this synthesis, two pore types are formed using only one P123 template. MCF-type cellular pores are surrounded by SBA-15-type mesopores. To our knowledge, there have been no reports on this type of hierarchical mesoporous– mesoporous material. Most hierarchical porous materials are macro–meso- or macro–microporous materials. HMMS was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), gas adsorption measurements, and small-angle X-ray scattering. A representative transmission electron microscopic image of HMMS (Figure 2 a) shows that 10-nm-sized ordered mesopores are associated with larger 40-nm-sized mesocellular pores. The cellular pores in HMMS are clearly shown in the scanning electron microscopic image (Figure 2 b). The TEM image (Figure 2 c), obtained by thin-sectioning a polymer-embedded sample, clearly shows that the large cellular pores of the HMMS are well-mixed with the smaller 10-nm-sized ordered mesopores. The structure of HMMS is a mixture of MCF[3] and SBA-15 silica[2a] in one mesostructured primary particle. Representative nitrogen adsorption/desorption isotherms and the corresponding pore-size distribution obtained from the analysis of the adsorption branch using the BJH (Barett–Joyner–Halenda) method are shown in Figure 3 a. The nitrogen isotherm
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Figure 2. a) Representative transmission electron microscopic (TEM) image of HMMS. b) Representative scanning electron microscopic (SEM) image of HMMS. c) TEM image of HMMS after microtoming.
shows two major capillary condensation steps at relatively high pressures of over 0.8 P/P0. The step at 0.8–0.9 P/P0 results from adsorption in the 10-nm-sized mesopores, while the other at 0.9–1.0 P/P0 is from adsorption in the 40-nmsized cellular mesopores. The presence of these two distinct types of pores is also clearly revealed in the pore-size distribution (PSD), which shows two peaks centered at 13.3 nm and 36.6 nm derived from the main mesopores and mesocellular pores, respectively. The BET surface area and singlepoint total pore volume of HMMS are 330 m2 g1 and 1.34 cm3 g1, respectively. The micropore volume, obtained by the Horvath–Kawazoe method based on a low-pressure isotherm, was about 10 % of the total pore volume of the HMMS silica. These micropores seem to have been generated as a result of the penetration of ethylene oxide (EO) groups into the silica walls.[4] From these characterizations, it is evident that HMMS is composed of 37 nm cellular mesopores and 13 nm ordered mesopores. Generally, only small mesopores (< 10 nm) exhibit an ordered structure in hierarchical mesostructured materials.
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Figure 4. Schematic representation showing the formation mechanism of HMMS.
Surprisingly, however, the small-angle X-ray scattering (SAXS) pattern of HMMS revealed the presence of ordered pore structures with two different length scales. Figure 3 b shows the SAXS spectrum obtained from HMMS, which shows two sets of scattering peaks that are indicated by the two closed circles at q = 0.21 and 0.42 nm1, and two asterisks at q = 0.55 and 0.97 nm1, where the first two peaks correspond to the reflections from the 30 nm pores, and the latter two peaks derive from the hexagonal (100) plane with an interplanar spacing (d) of 11.3 nm. The calculated values of the interplanar spacing (d100) and lattice parameter (a) from the small mesopores with a hexagonal phase are about 11.3 nm and 13 nm, respectively, while the larger cellular mesopores have a size of 31 nm. These results clearly demonstrate that the two different sets of pores are very uniform in size. When P123 was used as a structure-directing agent for assembling the mesostructure, only unimodal pores are generally obtained.[2a] To make mesocellular type pores, a pore expander such as trimethylbenzene is usually required.[3] However, in the synthesis of HMMS, mesocellular pores were generated without adding any pore expander. A plausible synthetic route for HMMS is schematically shown in Figure 4. In our synthesis, sodium silicate solution
is diluted with a large amount of water. When we used concentrated sodium silica instead of diluted sodium silicate, we obtained poorly defined silica materials. The pH value of both the undiluted and diluted sodium silicate solutions is over 14. Above pH 12, most silanols are known to be deprotonated and the major silica building blocks are present in the form of cyclic trimers and tetramers.[12] The interaction between P123 surfactant and the silica source is mainly based on hydrogen bonding under neutral conditions. The diluted sodium silicate can be neutralized faster than concentrated sodium silicate by acetic acid present in the synthetic medium. Thus the diluted sodium silicate interacts with the tri-block copolymers faster, resulting in the production of small-sized mesostructured building units. The residual P123 would be expected to interact with these preformed units, resulting in the formation of bilayer supermicelle structures.[7a, 13] The continuous cooperative synthesis of bilayer or globular structures on a silica surface was reported by Grant et al,[13] while Su and co-workers reported on hierarchical macro–mesoporous transition-metal oxide materials.[7a] They also used a single surfactant to make dual macro–mesoporous materials. The formation of hierarchical macro–mesoporous materials was explained by fast hydrolysis/condensation of transition metal alkoxides resulting in the formation of small mesostructured building units with a large number of hydroxyl groups, and the assembly of residual surfactants into multilayered micelles to make macropores. In our synthesis, prehydrolyzed sodium silicate was used instead of tetraethyl orthosilicate (TEOS). The pH value of our synthetic medium is 6.3–6.4 and the condensation rate is a maximum at around pH 6.[12] So, through a similar pathway, small-sized mesostructure silica building blocks with SBA-15 pores templated by P123 can be initially formed. Residual surfactants first adsorb onto the hydrophilic surface and self- assemble into large vesicle-type micelles, which are responsible for the formation of the mesocellular pores. In our study, the formation of a hierarchically ordered mesocellular mesoporous structure before hydrothermal treatment is corroborated by TEM and nitrogen isotherms (see Supporting Information). Hydrothermal treatment was conducted to make the silica walls more rigid. To demonstrate that the residual P123 surfactant plays a key role as a
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Figure 3. a) N2 adsorption/desorption isotherms of HMMS (Inset: The corresponding pore-size distribution). b) Small-angle X-ray scattering pattern of HMMS showing the regularity of large cellular pores and small ordered mesopores.
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of 3–4 nm.[16] In contrast, the current HMMC exhibited ordered mesopores along with the main cellular pores. The overall synthetic scheme for HMMC is presented in the Supporting Information. The TEM image of HMMC (Figure 6 a) shows that the 40 nm cellular pores of HMMS are well preserved and that small mesopores are also present. These small meso-
Figure 5. TEM images of mesoporous silica synthesized by using different amounts of P123 from the original recipe. a) When the amount of P123 was increased by 1.3 times, the resulting silica exhibited a hierarchical dual mesoporous structure consisting of large mesocellular pores and small mesopores. b) Reducing the amount of P123 to 0.7 times the original amount resulted in a unimodal silica material with a one-dimensional structure.
when the amount of P123 was reduced to only 0.7 times the standard amount, one-dimensional, hexagonally ordered silica with unimodal pores, similar to the SBA-15 structure, was obtained (Figure 5 b). Pinnavaia and co-workers reported the synthesis of ordered one-dimensional mesoporous molecular sieves (pore size 10 nm), denoted as MSU-H,[14] under near-neutral synthetic conditions. Although the synthetic method used for the fabrication of the HMMS is similar to that of MSU-H, HMMS possesses uniformly sized cellular mesopores along with 13-nm-sized mesopores, whereas no such cellular mesopores were observed in MSU-H.
2.2. Hierarchical Mesocellular Mesoporous Carbon and Polymer Materials from a HMMS Silica Template Uniform-sized mesoporous carbon materials formed using various mesostructured silica templates have attracted much attention for their possible application as electrode materials, adsorbents, and catalyst supports.[15] Recently, our group synthesized two kinds of bimodal mesoporous carbons using MCF, developed by Stucky and co-workers, and bimodal mesoporous silica, developed by our group, as inorganic templates.[16, 17] The key to the success of the preparation of these bimodal mesoporous carbons is the selective incorporation of carbon precursors. In the synthesis of mesocellular carbon foam, phenol vapor could not infiltrate into the large main cellular mesopores because a very high vapor pressure was required. Using a similar synthetic procedure and employing HMMS as the silica template, however, we were able to synthesize a new hierarchical mesocellular mesoporous carbon (HMMC). Although the synthesis of the mesocellular carbon foam with a bimodal pore structure has already been reported by our group, the small mesopores present in the walls were disordered pores with sizes
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Figure 6. a) Representative TEM image of HMMC. b) Representative SEM image of HMMC.
pores were generated by the replication of the 13 nm mesopores of the HMMS silica. The preservation of the large cellular pores is also confirmed by SEM (Figure 6 b). The individual particle size of HMMC is also a few hundred nanometers, which is favorable for the adsorption of large molecules when used as an adsorbent or as a nanometer-scale reactor for biomolecules.[16b] The N2 isotherms of HMMC (Figure 7 a) exhibited two major capillary condensation steps, resulting from the large cellular pores (P/P0 0.9) and small ordered mesopores (P/P0 0.6), respectively. The preservation of the ordered structure of the small pores contained in HMMS is attributed to the presence of the complementary pores present between the ordered small pores ( 13 nm).[4] The size of the pores generated from the dissolution of the silicate walls is 4.74 nm, as calculated using the adsorption isotherm based on the BJH method, which is somewhat larger than that of CMK-3[18] or C-MSU-H.[19] The pore-size distribution resulting from the large cellular pores ( 40 nm) is somewhat broadened compared with that of HMMS. The BET surface area and single-point total pore volume of HMMC are
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distribution of HMMP suggested 28-nm-sized mesocellular pores and 4-nm-sized ordered mesopores. The ordered structure of HMMP with its two different length scales is also clearly shown in the corresponding SAXS pattern. The SAXS spectrum presents two types of scattering peaks indicated by two closed circles and one asterisk at q = 0.18, 0.38, and 0.54 nm1, respectively. The peaks indicated by a closed circle represent cellular pores with a pore size of about 35 nm. The peak indicated by the asterisk represents the ordered small pores, which are generated by the negative replication of ordered small pores of HMMS silica. The BET surface area and single-point total pore volume of HMMP are 654 m2 g1 and 1.49 cm3 g1, respectively.
2.3. Application of HMMS Silica as a Host of Enzyme Immobilization
853 m2 g1 and 1.54 cm3 g1, respectively. The SAXS pattern of HMMC revealed that the regularity of both the large cellular pores and small mesopores of HMMS is preserved during the replication (Figure 7 b). The SAXS pattern shows two sets of scattering peaks. The peak indicated by the single closed circle results from the large mesocellular pores, while those indicated by the four asterisks are from the small ordered mesopores. The calculated values of the interplanar spacing (d100) and lattice parameter (a) of the small mesopores are about 10.0 nm and 11.7 nm, respectively. The peak indicated by the closed circle in Figure 7 b (q = 0.18 1) represents the mesocellular pores have a pore size of about 35 nm. Mesoporous polymers with interconnected large mesopores and high surface areas play a very important role as solid-phase supports for organic reactions that require bulky reagents.[20] A mesocellular mesoporous polymer foam composed of poly(divinylbenzene) with hierarchically ordered mesopores, here denoted as HMMP, was successfully synthesized using HMMS as a nanoscopic template. The hierarchically ordered structure of HMMS is successfully transferred to the HMMP, as confirmed by the N2 isotherms and SAXS pattern (see Supporting Information). The pore-size
Since the development of stable enzyme systems is of great importance for applications such as enzyme reactors, biosensors, and bioremediation, we have developed crosslinked enzyme aggregates (CLEAs) in HMMS, which can stabilize enzyme activity.[11, 21] The preparation of CLEAs in HMMS requires a two-step process. The first step involves the adsorption of enzymes into HMMS, which proceeds with a high degree of enzyme loading within a short time. The second step involves glutaraldehyde (GA) treatment, which results in the crosslinking of enzyme molecules to create aggregates within the pores of HMMS. This approach is designed to yield stable enzyme activity by preventing leaching, since the enzyme aggregates created in the larger mesocellular pores (37 nm) are not expected to leach out through the smaller mesoporous channels (13 nm). GA crosslinking is performed promptly after the enzyme adsorption, in order to enhance the high enzyme loading. HMMS can be shown to be a good host material for the synthesis of nanometer-sized enzyme aggregates for the following reasons. First, 13-nm-sized connecting mesopores are large enough for the facile passage of individual enzyme molecules with little diffusional limitation. This results in the quick adsorption of enzymes into HMMS. Second, the main 37-nm-sized spherical mesocellular pores can accommodate nanometer-sized crosslinked enzyme aggregates, which cannot leach out of HMMS through smaller mesoporous channels. This would result in a stable enzyme system as described below. To demonstrate the concept of CLEAs in HMMS, we prepared CLEAs containing a-chymotrypsin (CLEA–CT). While shaking (200 rpm), CT adsorption with a high loading of 36.3 wt % (570 mg of CT in 1 g of HMMS) was completed in less than five minutes. This rapid completion of high enzyme loading can be ascribed to both the small particle sizes and the good connectivity between the mesoporous channels (13 nm) and large mesocellular pores (37 nm). CT adsorption was followed by GA treatment and excessive washing for the preparation of CLEA–CT; the final CT loading in CLEA–CT was 33.2 wt %. In a control experiment without GA crosslinking, CT was leached from the HMMS during the incubation and washing steps, lowering
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Figure 7. a) N2 adsorption/desorption isotherms of HMMC (Inset: Corresponding pore-size distributions). b) Small-angle X-ray scattering pattern of HMMC showing the regularity of the large cells and small ordered pores.
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proach was successful in preventing enzyme aggregates in the main mesocellular pores (37 nm) from being leached through the bottlenecks of the mesoporous channels (13 nm). We also speculate that most of the CLEA–CT in the mesoporous channels are crosslinked with CLEAs in mesocellular pores and do not leach out from HMMS since the reduction of enzyme loading from adsorbed CT (36.3 wt %) to CLEA–CT (33.2 wt %) was marginal when compared to the volume fraction of mesoporous channels.[22] In addition, the observed stability indicates that CLEA–CT does not lose activity by autolysis due to the inhibition of autolysis after the enzymes are crosslinked. The kinetic constants of free CT, adsorbed CT, and CLEA–CT are shown in Table 1. The catalytic efficiency (kcat/Km) of CLEA–CT was 28 times lower than that of free CT, and it was due to both reduced kcat (17 %) and increased Km (4.8 times) values. The reduced kcat value can be explained by the reduced flexibility and deformation of CT enzyme molecules after being crosslinked. The increased Km value can be attributed to the increased mass-transfer limitation for the substrate in HMMS containing CLEA–CT. However, since a soluble form of the free enzymes cannot be recovered and recycled in real applications of enzymes, this lowered enzyme activity with CLEAs in HMMS is still useful in recycling enzymes and can be compensated by impressive stability and high enzyme loading, which makes it possible to recycle enzymes for more iterative uses and reduce the size of enzyme reactors. In addition, the catalytic efficiency (kcat/Km) of CLEA–CT was 10 times higher than Figure 8. Stability of free CT, adsorbed CT, and CLEA–CT under rigorthat of adsorbed CT due to higher kcat and lower Km values ous shaking (200 rpm). The relative activity (%) represents the ratio of residual activity to initial activity of each sample. of CLEA–CT. This is an additional advantage of CLEA–CT over adsorbed CT, together with better stability and higher enzyme loading. We speculate that the higher kcat value of porous silica were claimed to be stable, though only being CLEA–CT can be explained by the prevention of structural incubated under static conditions. For practical applications, deformation of the CT molecules, which can be serious with it is critical to preserve the initial activity under harsh condiadsorbed CT molecules due to denaturation and/or autolytions, since most reactions using immobilized enzymes are sis. Interestingly, adsorbed CT with lower CT loading conducted under shaking. Adsorbed CT with no GA treat(6.8 wt %) has a higher Km value than CLEA–CT with ment showed a continuous loss of CT activity, and the leaching of CT was confirmed by measuring the amount of CT in higher loading (33.2 wt %). This suggests that adsorbed CT the supernatant. In contrast, CT activity was clearly stabiplaces more serious mass-transfer limitations on the sublized when the samples were treated with 0.1 % GA. This strate than CLEA–CT, even though the internal porosity of CLEA–CT showed no decrease in CT activity throughout a HMMS with adsorbed CT contains smaller amounts of CT two-week incubation period. The half-lives of free and admolecules than that with CLEA–CT. This puzzling result sorbed CT were calculated to be 1 h and 3.6 days, respeccan be explained by the denaturation and/or autolysis of adtively. On the other hand, CLEA–CT did not show any acsorbed CT molecules, which leads to an increase of their octivity loss even during extended incubation up to one month cupied volume and more serious mass-transfer limitation for (data not shown). This impressive stability under rigorous substrates in HMMS. CLEA–CT would not have this kind conditions demonstrates that the “ship-in-a-bottle” apof problem since multi-point attachments in the form of CLEAs would prevent the denaturation of CT mole[a] Table 1. CT loading and kinetic constants of free CT, adsorbed CT, and CLEA–CT. cules. We can even anticiSamples CT Loading (wt%) kcat [s1] Km [mm] kcat/Km [ 103 m1 s1] pate a small degree of volume shrinkage during Adsorbed CT 6.8 0.8 0.1 291 24 2.7 0.3 crosslinking and more conCLEA–CT 33.2 5.2 0.4 186 23 28 4 Free CT 29.9 0.7 39 3 770 6 trolled distribution of CLEA–CT in HMMS. In [a] The CT activity was determined by the hydrolysis of TP (1.6–160 mm) in an aqueous buffer (10 mm other words, adsorbed CT phosphate, pH 7.8) at room temperature (22 8C). The active-site concentrations were determined by the does not have any control MUTMAC assay.[25] Kinetic constants were obtained by using software (Enzyme Kinetics Pro from over the distribution of ChemSW, Farifield, CA) that performs nonlinear regression based on the least-squares method. the final loading to only 6.8 wt %. This demonstrates the value of the GA treatment for capturing high enzyme loadings. Figure 8 shows the stability of free CT, adsorbed CT, and CLEA–CT in aqueous buffer (10 mm sodium phosphate, pH 7.8) at room temperature. The samples were shaken side-by-side (horizontally) at 200 rpm. These rigorous conditions for testing stability have not been used in most other studies,[8, 9] where the adsorbed enzymes in meso-
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enzyme molecules by nature, and small mesoporous channels of HMMS may be continuously filled with intact, denatured, and autolyzed CT molecules, leading to a more detrimental mass-transfer limitation than seen for CLEA–CT.
3. Conclusions In conclusion, hierarchically ordered mesocellular mesoporous silica (HMMS) materials were synthesized using a single structure-directing agent under neutral conditions. As described above, most of the hierarchical porous materials reported so far are meso–macroporous or micro–macroporous materials. In addition, HMMS have two advantages over other mesoporous materials. First, the overall synthetic process is very cost-effective because inexpensive sodium silicate was employed as the silica source and the synthesis is conducted under mild, neutral conditions. Secondly, the synthetic procedure using a single template is much simpler than those employed for the synthesis of other hierarchically ordered mesoporous materials. Using HMMS as a nanoscopic template, hierarchically ordered mesocellular mesoporous carbon and polymer materials were successfully synthesized. Such silica, carbon, and polymer materials have the potential to be used as a host of catalysts and with large-sized molecules such as biomolecules. We also developed immobilized enzyme reactors in the nanometer-scale pores of a bimodal HMMS. Our approach using glutaraldehyde for the purpose of crosslinking captures very high enzyme loadings and prevents leaching, providing a more stable and active immobilized enzyme system than those obtained by simple adsorption. Thus, we have developed a “ship-in-a-bottle” approach to obtain active and stable enzyme reactors in the pores of a uniquely designed mesoporous material (HMMS). For example, CLEAs containing a-chymotrypsin (CLEA–CT) did not show any activity decrease under rigorous shaking for one month, which demonstrates a huge success of this approach. This stable and active enzyme system is expected to make a broad impact in various enzyme applications such as bioremediation, biosensors, and bioconversion.
4. Experimental Section Synthesis of hierarchically mesocellular mesoporous silica (HMMS): 9.7 g of P123 ((EO)20(PO)70(EO)20) and 4.48 mL of concentrated acetic acid were dissolved in 200 mL of water. The resulting solution was heated to 60 8C and maintained at that temperature for 1 h. Then, 16 mL of sodium silicate diluted with 200 mL of water was poured into the solution with vigorous stirring. On mixing the two solutions, the temperature was dropped to 45–47 8C. The pH value of the reaction mixture was 6.3–6.4. The molar composition of the synthetic mixture was SiO2 :P123:acetic acid:H2O = 1:0.0167:0.078:222.2. The solution was reheated to 60 8C and aged at that temperature for 20 h, folsmall 2005, 1, No. 7, 744 –753
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lowed by hydrothermal treatment at 100 8C for 24 h. Calcination of the filtered materials at 550 8C generated HMMS. Synthesis of HMMC: The alumination (Si/Al = 20) of pure silica HMMS was performed by means of the impregnation method, in order to generate acidic catalytic sites. 1.3 mL phenol per gram of HMMS was incorporated into the pores of the HMMS, by heating a mixture of HMMS and phenol at 140 8C under a static vacuum. The resulting phenol-incorporated HMMS and formaldehyde were reacted in an autoclave at 130 8C for 2 days inside the pores of the HMMS to yield the phenol resin/HMMS nanocomposite. The nanocomposite was heated to 160 8C at 1 8C min1 and held at this temperature for 5 h under flowing nitrogen. The temperature was then ramped at 5 8C min1 to 850 8C and held at this temperature for 7 h to carbonize the phenol resin inside the pores of the HMMS, so as to obtain the carbon/ HMMS nanocomposite. The dissolution of HMMS using 1 m NaOH in a 1:1 mixture of EtOH and H2O generated HMMC. Synthesis of HMMP: The calcined HMMS was dehydrated at 200 8C under vacuum for 4 h. A polymer precursor solution composed of divinylbenzene and 2,2’-azobisisobutyronitrile (AIBN; 15:1) was wetted into the pores of the HMMS silica using the incipient wetness method. The amount of divinylbenzene was adjusted to 50 % of the pore volume of the HMMS template, in order to preserve the cellular structure. Polymerization was performed by heating at 85 8C for 24 h under an argon atmosphere. Removal of the silica template using 10 wt % HF diluted with ethanol yielded HMMP. Enzyme immobilization in HMMS: HMMS (10 mg) was mixed with 1.5 mL of 4 mg mL1 free CT in a buffer solution (10 mm sodium phosphate buffer, pH 7.8), vortexed for 30 s, sonicated for 3 s, and incubated at room temperature while shaking (200 rpm). After 20 min incubation for the adsorption of free CT in HMMS, the samples were washed very briefly in sodium phosphate buffer (100 mm sodium phosphate, pH 8.0), and incubated with 0.1 % glutaraldehyde solution in phosphate buffer (100 mm sodium phosphate buffer, pH 8.0) at 200 rpm for 30 min. After GA treatment, the samples were washed by phosphate buffer (100 mm sodium phosphate, pH 8.0) and Tris-HCl buffer (100 mm Tris, pH 8.0), respectively. The capping of unreacted aldehyde groups was performed in a fresh Tris-HCl buffer (100 mm Tris, pH 8.0) at 200 rpm for 30 min. After Tris-capping, the samples were washed two times by phosphate buffer (10 mm sodium phosphate, pH 7.8), and stored at 4 8C. The adsorbed CT was also prepared by using no GA during the treatment process, but the washing was performed in the exactly same way as for CLEA–CT. Protein leaching from HMMS into the supernatant was measured by the BCA method[23] at each washing step together with control samples, and used for the calculation of final CT loading. Activity and stability of CLEA–CT: The activity of the immobilized CT was determined by the hydrolysis of 160 mm N-Succinyl-AlaAla-Pro-Phe p-nitroanilide (TP) in an aqueous buffer (10 mm sodium phosphate, pH 7.8) at room temperature. After 30 s of vortexing, the samples were shaken at 250 rpm, and the increase in absorbance at 410 nm in the supernatant was mea 2005 Wiley-VCH Verlag GmbH & Co. KGaA, D-69451 Weinheim
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full papers sured time-dependently after centrifugation of the suspension at 5000 G. The activity measurement was performed within 20 to 30 min to reduce the possible effect of leached enzymes on the activity results. The stability of CLEA–CT was checked in aqueous buffer (10 mm sodium phosphate, pH 7.8) at room temperature under shaking (200 rpm). At each time point, an aliquot of each sample was added to the aqueous buffer containing TP, and the residual activity was measured as described above. The relative activity (%) was calculated from the ratio of the residual activity to the initial activity of each sample. Characterization: Transmission electron microscopic images were obtained on a JEOL EM-2010 microscope. Scanning electron microscopic images were obtained on a JSM-840A microscope. N2 adsorption/desorption isotherms at 77 K were obtained using a Micromeritics ASAP2010 sorptometer. Pore-size distributions were calculated using the BJH (Barett–Joyner–Halenda) method. Synchrotron SAXS measurements were performed on the 4C2 Beamline at the Pohang Light Source (Korea). The primary beam was monochromatized with a coupled Si(111) single crystal at a wavelength of 0.1608 nm (the photon energy of the X-ray is 7.78 keV, a resolution Dl/lffi0.0001), and then it was focused on a detector plane by means of a bent cylindrical mirror. A 2D CCD camera (Roper Scientific Inc., PI-SCX-2048) was used to collect the scattered X-rays. We used the SEBS block copolymer (32.5 nm d spacing) as a periodic calibrant, in order to calibrate the image from the 2D CCD camera.[24]
J. Kim, T. Hyeon, et al.
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Acknowledgments T.H. is grateful for financial support from the Korean Ministry of Science and Technology through the National Creative Research Initiative Program. J.K. would like to thank U.S. Department of Energy (DOE) LDRD funds administered by the Pacific Northwest National Laboratory, and the DOE Office of Biological and Environmental Research under the Environmental Management Science Program. The research was performed in part at the W. R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. We thank Prof. Chae-Ho Shin at the Chungbuk National University for the micropore structure characterization. We also thank Dr. Hyunmin Park at the Korea Research Institute of Standards and Science for the small-angle X-ray scattering studies.
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