Highly Efficient Enzyme Immobilization and Stabilization within Meso

Jan 31, 2012 - Hyunmin Park,. ⊥. Jong Ho Lee,. †. Sang-Mok Lee,. #. Dohoon Lee,. ▽. Sangyong Kim,. ▽. Yoon-Mo Koo,. #. Chae Ho Shin,. ○. Seu...
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Highly Efficient Enzyme Immobilization and Stabilization within Meso-Structured Onion-Like Silica for Biodiesel Production Seung-Hyun Jun,† Jinwoo Lee,*,‡ Byoung Chan Kim,§ Ji Eun Lee,◆ Jin Joo,∥ Hyunmin Park,⊥ Jong Ho Lee,† Sang-Mok Lee,# Dohoon Lee,▽ Sangyong Kim,▽ Yoon-Mo Koo,# Chae Ho Shin,○ Seung Wook Kim,† Taeghwan Hyeon,*,◆ and Jungbae Kim*,† †

Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Korea Advanced Functional Nanomaterials Laboratory, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Korea § Environment Sensor System Research Center, Korea Institute of Science and Technology, Seoul 136-791, Korea ∥ Department of Applied Chemistry, Kyungpook National University, Daegu, Korea ⊥ Material Evaluation Center, Korea Research Institute of Standards and Science, Taejon 305-600, Korea # Center for the Advanced Bioseparation Technology, Inha University, Incheon 402-751 Korea ▽ Green Process R&D Department, Korea Institute of Industrial Technology, Cheonan, Chungnam 331-825, Korea ○ Department of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk 360-763, Korea ◆ National Creative Research Initiative Center for Oxide Nanocrystalline Materials and School of Chemical Engineering, Seoul National University, Seoul 151-744, Korea ‡

S Supporting Information *

ABSTRACT: Meso-structured onion-like silica (Meso-Onion-S) was synthesized and used as a host of enzyme immobilization. Meso-Onion-S has a 200− 300 nm sized primary meso-structured onion building unit, and each onion unit has highly curved mesopores of 10 nm diameter in a multishell structure. Nanoscale enzyme reactors (NERs) in Meso-Onion-S were prepared via a twostep process of enzyme adsorption and subsequent enzyme cross-linking, which effectively prevents the leaching of cross-linked enzyme aggregates from highly curved mesopores of Meso-Onion-S. As a result, NERs in Meso-Onion-S significantly improved the enzyme stability as well as the enzyme loading. For example, NER of lipase (NER-LP) was stable under rigorous shaking for 40 days, while the control sample of adsorbed LP (ADS-LP) with no enzyme cross-linking showed a rapid inactivation due to rigorous enzyme leaching under shaking. Stable NER-LP was successfully employed to produce biodiesels and fatty acid methyl esters, from the LP-catalyzed transesterification of soybean oil with methanol. Interestingly, the specific activity of NERLP was 23 and 10 times higher than those of free LP and ADS-LP, respectively, revealing the importance of LP stabilization in the form of NER-LP in the presence of organic solvents. KEYWORDS: meso-structured onion-like silica, enzyme immobilization, enzyme stabilization, biodiesel production

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INTRODUCTION Nanobiocatalysis, incorporating enzymes into nanomaterials or synthesizing the hybrid materials of enzymes and nanomaterials, is gathering more and more attention due to recent successes in stabilizing the enzyme activities.1,2 Especially, mesoporous materials have attracted much attention because of their controlled porosity and high surface areas.3−10 A simple adsorption method, due to its simplicity, is most frequently used to immobilize enzymes into mesoporous materials3−9 but has a serious problem of enzyme leaching. To prevent the leaching of enzymes, enzyme molecules were covalently attached in mesoporous materials,10 but this approach uses only the inner surface of mesopores rather than the whole pore © 2012 American Chemical Society

volume that can lead to poor enzyme loadings. Another unique approach is to let enzymes adsorb into mesoporous silica and encapsulate the enzyme-immobilized mesoporous silica by assembling a multilayered nanocomposite shell on the surface of mesoporous silica particles.11,12 This approach resulted in high enzyme loadings and stability, but the outer shell intrinsically placed an additional limitation against the transfer of substrate and products. As an approach to improve both Special Issue: Materials for Biological Applications Received: July 25, 2011 Revised: January 29, 2012 Published: January 31, 2012 924

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oryzae, and glutaraldehyde (GA) and 4-nitrophenyl butyrate (4-NB) were purchased from Sigma (St. Louis, MO, USA). Soybean oil was purchased from Yakuri Pure Chemicals (Kyoto, Japan). Methyl alcohol was purchased from Carlo Erba (Milano, Italy). The BCA (bicinchoninic acid) protein assay reagents were purchased from Pierce (Rockford, IL, USA). Isooctane was purchased from Daejung Chemicals (Siheung, Korea). Synthesis of Meso-Onion-S. The typical synthetic procedure for Meso-Onion-S is as follows. A total of 4 g of P123 was dissolved in 150 mL of 1.6 M HCl at 20 °C. A total of 2 g of TMB was added and stirred further for 5 h at room temperature. Then, the solution was heated to 40 °C and 8.5 g TEOS was added under rigorous stirring. The solution was aged at 40 °C for 20 h under magnetic stirring and further aged at 100 °C in a polypropylene bottle with the cap closed under static condition for one day. The product was filtered and calcined at 550 °C for 4 h to generate Meso-Onion-S. The aging process was performed at different time spans for the synthesis of Meso-Onion-S-X, where X indicates the hydrothermal treatment time at 100 °C. Enzyme Immobilization in Meso-Onion-S. Meso-Onion-S (100 mg) was mixed with 2 mL of 100 mg mL−1 crude lipase solution (20 mM sodium phosphate buffer, pH 7.0) in a centrifuge tube and incubated under shaking at 200 rpm with the tube in the upright position. The amount of adsorbed lipase into Meso-Onion-S was monitored time-dependently by measuring the enzyme amount in the solution at each time point and calculating the disappeared enzyme amount as the loading of adsorbed lipase into Meso-Onion-S. The BCA protein assay was used to determine the enzyme concentration in an aqueous solution.23 For the synthesis of NER-LP, adsorbed lipase in Meso-Onion-S was washed very briefly by using phosphate buffer (100 mM sodium phosphate, pH 7.0) and incubated in 0.5% glutaraldehyde solution at 200 rpm for 1 h. After the GA treatment, the samples were washed by phosphate buffer (100 mM sodium phosphate, pH 7.0) and Tris-HCl buffer (100 mM Tris, pH 7.0), respectively. The capping of unreacted aldehyde groups was performed in a fresh Tris-HCl buffer (100 mM Tris, pH 7.0) at 200 rpm for 30 min. After Tris-capping, the samples were washed five times by using phosphate buffer (20 mM sodium phosphate, pH 7.0) for 5 min and stored in 20 mM sodium phosphate buffer (pH 7.0) at 4 °C until use. The control samples of adsorbed lipase in Meso-Onion-S (ADS-LP) were also prepared by using a buffer solution with no GA during the GA treatment process for NER-LP, but the washing was performed in the exactly same way as that for NER-LP. As a direct measurement of enzyme loading, an Elemental Analyzer (Vario EL III, Elementar Analysensysteme GMBH, Hanau, Germany) was used to obtain the nitrogen content, which enables the calculation of enzyme amounts in the samples because the enzymes are the only source for the measured nitrogen amount. In the case of NER-LP, Tris-capping was not performed to prevent the contribution of Tris to the total amount of nitrogen in the elemental analysis. Activity and Stability Measurements. The activity of immobilized lipase was determined by the hydrolysis of 40 μM 4nitrophenyl butyrate (4-NB) in an aqueous buffer (20 mM sodium phosphate, pH 7.0) at room temperature. The formation of a product, p-nitrophenol, was monitored by the increase of absorbance at 400 nm (A400) using a UV−vis spectrophotometer from Shimadzu (Kyoto, Japan). The enzyme activity was calculated by the slope of timedependent increase of A400. The enzyme stability in an aqueous buffer was determined by checking the residual activity after incubation of samples under rigorous shaking (200 rpm, side by side) at room temperature. After each measurement of residual activity, the samples were washed three times using an aqueous buffer to remove the substrate and products from the samples. Excessively washed samples were incubated under rigorous shaking and reused for the next measurement of residual activity. The relative activity at each time point was calculated from the ratio of residual activity to the initial activity of each sample. Biodiesel Production. The transesterification reaction was performed in a glass vial containing soybean oil (0.2 mmol), methanol (0.3 mmol), and 30−125 mg of crude or immobilized lipase in 10 mL

enzyme loading and stability without an outer shell, we reported a ship-in-a-bottle approach under the name of nanoscale enzyme reactors (NERs), which consists of enzyme adsorption into mesoporous materials and subsequent enzyme cross-linking.13 This NER approach has demonstrated its effective stabilization of various enzymes13−15 and potential applications in magnetically separable hybrids,16,17 magnetoelectrobiocatalysis,18 and enzyme-linked immunosorbent assay.18,19 Most cases of enzyme stabilization in a form of NERs were realized via a ship-in-a-bottle mechanism by employing mesoporous materials with a bottleneck pore structure, which effectively prevents the leaching of crosslinked enzyme aggregates in larger mesocellular pores through smaller connecting mesopores.13,14,16−20 The resulting improvement of operational enzyme stability, together with the prevention of enzyme denaturation due to multipoint covalent linkages on the surface of enzyme molecules, has resulted in highly stable NERs as well as high enzyme loadings.13,14 Interestingly, the NER approach could also make successful enzyme stabilization in SBA-15, which has linear mesoporous channels with no bottleneck pore structure. The effective enzyme stabilization was explained by the bent pore structure of SBA-15 with a high aspect ratio, which can effectively prevent the leaching of one-dimensional cross-linked enzyme aggregates from the mesoporous channels of SBA-15.15 In the present work, we synthesized mesoporous vesicle silica with large pores (∼10 nm) and hierarchical structures by using 1,3,5-trimethylbenzene (TMB) as a modifier of the surfactant packing parameter (Supporting Information). We used the same chemical composition for the synthesis of mesocellular siliceous foam (MCF) with a cell and window mesostructure21,22 but slightly changed the synthetic condition by extending the stabilization time of TMB at room temperature for the preparation of mesoporous vesicle silica, called “mesostructured onion-like silica (Meso-Onion-S)”. Meso-Onion-S was employed for the preparation of NERs with lipase (NERLP). LP was quickly adsorbed into Meso-Onion-S due to the smaller size of the onion building unit (200−300 nm), but the adsorbed lipase without the step of enzyme cross-linking (ADSLP) leached out very rapidly under shaking condition. However, a simple additional step of enzyme cross-linking after enzyme adsorption resulted in improved enzyme stability due to a highly curved pore structure of Meso-Onion-S, which effectively prevents the leaching of cross-linked enzyme aggregates even under rigorous shaking. To demonstrate a potential application of highly stable NER-LP in Meso-OnionS, the LP-catalyzed transesterification of soybean oil with methanol was performed to generate the biodiesels in the form of fatty acid methyl esters. This is the first demonstration of NER-LP uses for the production of biodiesels in organic solvents. To our surprise, the high stability of NER-LP in organic solvents allowed for high specific activity of NER-LP, which was 23 and 10 times higher than those of free LP and ADS-LP, respectively.

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EXPERIMENTAL SECTION

Chemicals and Materials. Poly(ethlyene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) triblock copolymer EO20PO70EO20, denoted P123 (M n = 5800), and tetraethyl orthosilicate (TEOS) were purchased from Aldrich (Milwaukee, WI, USA). 1,3,5-Trimethylbenzene (TMB, 98%) was purchased from Merck (Darmstadt, Germany). Hydrochloric acid was purchased from Samchun Pure Chemical (Pyeongtaek, Korea). Lipase from Rhizopus 925

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of isooctane. To dehydrate the enzymes, crude and immobilized lipases were lyophilized by using a freeze-dryer (FDU-2100 and DRC1000, Eyela, Tokyo, Japan) for 24 h. The reaction mixture was incubated under shaking (250 rpm) at 45 °C. At each time point, an aliquot (100 uL) was withdrawn from the reaction mixture and centrifuged. Then, the supernatant solution was put into a dry bath at 90 °C for 15 min to kill the activity of lipases via thermal denaturation. Methyl ester contents were analyzed by using a capillary column (id 0.25 mm, length 30 m; HP-INNOWAX, Agilent, Santa Clara, CA, USA) in a M600D gas chromatography (Younglin Co., Anyang, Korea). The injector and flame ionization detector (FID) temperatures were both 260 °C. The oven temperature was increased from 150 to 180 °C at a rate of 15 °C/min, then increased to 240 °C at a rate of 5 °C/min, and held at 240 °C for 1 min. The retention times of palmitic acid methyl ester, stearic acid methyl ester, oleic acid methyl ester, linoleic acid methyl ester, and linolenic acid methyl ester were 3.6, 5.3, 5.5, 6.0, and 6.7 min, respectively. The conversion was calculated from the total amount of produced fatty acid methyl esters.

The critical synthetic parameter for Meso-Onion-S was to mix TMB with P123 for more than 5 h at room temperature. Although TMB was also mixed with P123 during the synthesis of MCF, excess amount of TMB led to the core oil droplets, which resulted in mesocellular siliceous foam with spherical large-sized pores.24 However, a long time stabilization of TMB with P123 at room temperature changed the surfactant packing factor rather than forming core micelles via more intimate mixing of TMB with P123 (Figure 1). The surfactant packing factor (P) is defined by the ratio of the effective cross-sectional area of the tail (hydrophobic) group (aT) to that of the head (hydrophilic) group (aH).25 When most of TMB interfaced with the surfactant tail group rather than forming core oil droplets, the P value would be increased to more than 1, which resulted in lamellar structures of Meso-Onion-S. This is an interesting finding because completely different mesostructured materials were synthesized using the same composition of starting materials when the synthetic condition was slightly changed to modify surfactant packing factors. Figure 1 also shows the transmission electron microscope (TEM) images of Meso-Onion-S and MCF silica. Meso-OnionS is comprised of 200−300 nm sized onion building units. Each onion has well-developed 10 nm sized mesopores with multishell structures, and the particle size of onion is relatively uniform compared to the macroscopic size of MSU-G.26 The scanning electron microscopic (SEM) image (see Supporting Information Figure S1) shows that a few hundred nanometer sized onions are aggregated into secondary micrometer sized particles. The structure of Meso-Onion-S is very similar to MLV (multilayer vesicular structures) synthesized using poly(ethylene oxide)−poly(butylene oxide)−poly(ethylene oxide) (PEO-PBO-PEO) surfactant.27 However, the present work describes the synthesis of onion-like mesostructure by modifying the surfactant packing parameter using the same composition for the synthesis of MCF.21,22 The samples of Meso-Onion-S, hydrothermally treated for 2, 3, 5, and 8 days, had the same layered structure (see Supporting Information Figures S2, S3, and S4), suggesting that the layered structure of Meso-Onion-S is not a metastable intermediate structure but a thermodynamically stable structure.28 The preservation of the layered structure, after the removal of structure directing agent (P123), suggests that there are some pillaring silicates between silica layers, which was observed in MSU-G silica.26 The Pinnavaia group proposed that the vesicle-like structure has fully accessible open pores, which was supported by the TEM images together with high catalytic activity of Al-MSU-G for the conversion of 2,4ditertbutylphenol (DTBP).29 Small angle X-ray scattering data reveals that the Meso-Onion-S has highly ordered layered structure (Figure 2a). Two (100) and (200) peaks are clearly shown, which is typical for the layered structure. The spacing value between the layers is 11.54 nm. The N2 isotherms of Meso-Onion-S at high relative pressure (>0.7 P/P0) have two adsorption steps (Figure 2b). The very steep one at P/P0 = 0.8 is derived from the uniform ∼10 nm pores between layers, and the other one might be from the interparticle pores between the onion structures. Pore size distribution calculated from the adsorption branch is very narrow at 9.5 nm. The pore size is similar to or slightly larger than that of SBA-15 synthesized by using only the P123 surfactant,30 explaining that TMB acted only as a modifier of the surfactant packing factor. By increasing the hydrothermal reaction time from 2 to 8 days, the pore size of Meso-Onion-S was increased from 9.5 to 11.0 nm

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RESULTS AND DISCUSSION Synthesis and Characterization of Meso-Onion-S. Meso-Onion-S with large-sized pores and highly curved structure was synthesized by modifying a surfactant packing parameter of the synthetic template solution. Figure 1 shows a

Figure 1. Schematic diagram showing the formation of MCF and Meso-Onion-S. During the MCF synthesis, oil (TMB) first swells out the PPO chain part and pure oil cores were formed.22 During the synthesis of Meso-Onion-S, TMB was mixed with P123 for more than 5 h at room temperature, which resulted in a good interfacing of TMB with the PPO part rather than forming core droplets. The surfactant packing factor was increased (P > 1), which is responsible for the formation of layered structure in Meso-Onion-S.

schematic for the difference in the syntheses of Meso-Onion-S and MCF. By mixing TMB with P123 surfactant ((EO)20(PO)70(EO)20) for a longer period of time such as more than 5 h, we could obtain mesoporous silica with onionlike structure (Meso-Onion-S) instead of MCF with a cell and window mesostructure. In the synthesis of MCF silica,21,22 TMB in a weight ratio to P123 larger than 30% was used as a pore expander to make large sized mesocellular pores (>20 nm) interconnected with window mesopores. In the present work, however, TMB was used as a modifier of surfactant packing parameter (P > 1) in the template solution by employing a longer time of mixing, which resulted in Meso-Onion-S with 10 nm mesoporous channels with no window mesopores. 926

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Figure 2. (a) Small-angle X-ray scattering pattern of Meso-Onion-S. (b) N2 adsorption−desorption isotherms and corresponding pore size distribution obtained from the adsorption isotherm.

(Supporting Information Figure S3). This can be explained by the increased hydrophobicity of the poly(ethylene oxide) group at high temperature.30 The BET surface area and pore volume measured at P/P0 = 0.99 are 673 m2/g and 1.1 cm3/g, respectively. The micropore volume of Meso-Onion-S measured at P/P0 = 0.1 is 0.243 cm3/g. These micropores were formed by the penetration of the ethylene oxide group in P123 into the silicate sheets between layers.31 The pore structure of Meso-Onion-S is very similar to that of vesicular or onion type mesoporous materials, reported by Pinnavaia group, except that the pores in Meso-Onion-S are larger than 9 nm.29,32 From a TEM image provided in the Supporting Information (Figure S5), some parts of the onion mesochannels are exposed to outer surfaces. Loading and Activity of Lipase in Meso-Onion-S. Lipase from Rhizopus oryzae was quickly adsorbed into 10 nm mesopores of Meso-Onion-S. Within 10 min, for example, Meso-Onion-S was saturated with adsorbed lipase in an enzyme loading of 33 ± 3.4% even under the mildest shaking condition to be tested (200 rpm and vial in an upright position). The quick adsorption and high loading of LP into Meso-Onion-S can be explained by a small-sized individual onion (200−300 nm) and well-exposed inlets of 10 nm mesopores to the exterior. Meso-Onion-S was used for the preparation of ADS-LP and NER-LP (Figure 3). After excessive washings until no detectable lipase in the washing solution, the enzyme loading of ADS-LP was 25 ± 2.5% (w/w). The enzyme loading of NERs was difficult to obtain due to the formation of insoluble cross-linked LP aggregates; it was 30 ± 1.0% (w/w) via elemental analysis to directly determine the enzyme loading from the measured amount of nitrogen, respectively. This reveals the effect of enzyme cross-linking in the form of NERLP that prevents the leaching of lipase from Meso-Onion-S. We reported that even a mildly bent structure of mesoporous channels in SBA-15 makes it difficult for one-dimensional crosslinked enzyme aggregates to be leached out of SBA-15.15 The additional simple step of enzyme cross-linking would generate one-dimensional cross-linked enzyme aggregates in highly curved mesopores of Meso-Onion-S, which would place a stronger resistance against the leaching of enzymes from MesoOnion-S. The NER approach, consisting of enzyme adsorption and cross-linking steps, is proven to effectively prevent the leaching of enzymes from Meso-Onion-S. The specific activities of free LP, ADS-LP, and NER-LP in the 4-NB hydrolysis were 110, 38.8, and 34.5 μM/min per mg LP, respectively. This result suggests that the specific activities of ADS-LP and NER-LP represent 35% and 31% of free LP,

Figure 3. Preparation of nanoscale enzyme reactor (NER) in MesoOnion-S and the enzyme leaching of adsorbed enzymes (ADS) under rigorous shaking (200 rpm) but not with NER.

respectively. These lowered specific activities of ADS-LP and NER-LP would be due to the mass transfer limitation while the lower specific activity of NER-LP than that of ADS-LP can be explained by the deformed enzyme conformation and active sites during chemical enzyme cross-linking. For comparison, ADS-LP and NER-LP were prepared by using MCF and SBA-15 as a host of enzyme immobilization, and their activities were measured by the hydrolysis of 4-NB. The specific activities of ADS-LP in MCF and SBA-1533 were 19.3 and 9.10 μM/min per mg LP, respectively. This suggests that the specific activity of ADS-LP in Meso-Onion-S was 2.1 and 4.3 times higher than those of ADS-LP in MCF and SBA15, respectively. The activity data of NER-LP also showed a similar trend with the specific activity of NER-LP in MesoOnion-S to be 1.6 and 5.1 times higher than those of NER-LP in MCF and SBA-15, respectively. Higher specific activities of immobilized LP in Meso-Onion-S can be potentially explained by the lower mass transfer limitation, when compared with MCF and SBA-15, which can be determined by several factors such as unit particle size, pore structure, and interpore connectivity. The unit particle size of Meso-Onion-S (200− 300 nm) is smaller than those of MCF (5−10 μm) and SBA-15 (1−2 μm). MCF has mesocellular pores (23 nm) well connected with one another via window pores (9.7 nm), while the linear mesopores with high aspect ratio of MesoOnion-S (10 nm) and SBA-15 (8.6 nm) are not well connected with one another (Supporting Information Table S1). To check the mass transfer limitation, the apparent Km value of each enzyme immobilization was obtained and summarized in Supporting Information Table S2. The specific activities showed a good linear correlation with the inverse values of Km (Supporting Information Figure S6), supporting the hypothesis that the higher specific activities of immobilized LP in Meso-Onion-S are due to lower mass transfer limitation. Stability of ADS-LP and NER-LP in Meso-Onion-S. Figure 4 shows the stabilities of ADS-LP and NER-LP in an aqueous solution under rigorous shaking (200 rpm). The LP activity was measured by the hydrolysis of 4-NB in an aqueous solution (20 mM sodium phosphate, pH 7.0), and the relative activity at each time point was calculated from the ratio of residual activity to the initial activity of each sample. ADS-LP 927

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phase, the labile form would contribute to the increase of product formation, but not in the second phase because the labile form would be inactivated during the first phase.35 The initial transesterification rates of ADS-LP and NER-LP in the first phase were 0.23 and 1.96 mM/min per 100 mg of MesoOnion-S, respectively. This suggests that the initial activity of NER-LP was 8.5 times higher than that of ADS-LP. It is generally accepted that lipase is denatured and loses its active conformation in the presence of methanol during the biodiesel production.36 Then, the higher activity of NER-LP can be explained by the better stability of NER-LP due to the prevention of enzyme leaching and denaturation as a result of a simple enzyme cross-linking step. The transesterification rates of ADS-LP and NER-LP in the second phase were reduced to 0.052 and 0.62 mM/min per 100 mg of Meso-Onion-S, respectively, suggesting that the activity of NER-LP was 12 times higher than that of ADS-LP. This result suggests more rigorous inactivation of ADS-LP than NER-LP during the transition from the first phase to the second phase. The specific activities of free lipase, ADS-LP, and NER-LP in the second phase of transesterification reaction in an organic solvent were 0.916, 2.16, and 21.3 μM/min per mg of LP, respectively. This suggests that the specific activity of NER-LP was 23 and 10 times higher than those of free LP and ADS-LP, respectively. For the case of 4-NB hydrolysis in an aqueous buffer, the specific activities of free LP, ADS-LP, and NER-LP were 110, 38.8, and 34.5 μM/min per mg of LP, respectively. In other words, the immobilized lipases in Meso-Onion-S, retaining poorer activity than free LP in an aqueous buffer due to mass transfer limitation, showed higher activity than free LP for the case of transesterification reaction in an organic solvents. Then, the higher specific activity of ADS-LP than free lipase in the biodiesel production can be explained by both the protection of lipase in nanoconfined mesopores of MesoOnion-S7 and the activation of lipase in Meso-Onion-S via a lidopening mechanism.37 Further enzyme stabilization in the form of NER-LP, preventing the leaching and denaturation of lipase in the presence of organic solvents, can explain the 23 and 10 times higher activity of NER-LP than those of free LP and ADS-LP, respectively.

Figure 4. Stabilities of ADS-LP and NER-LP under rigorous shaking (200 rpm). The relative activity at each time point represents the ratio of residual activity to the initial activity of each sample.

showed a rapid decrease of LP activity due to the leaching of LP from Meso-Onion-S and follow-up denaturation of LP under rigorous shaking. On the other hand, NER-LP showed a good stabilization of LP activity, which can be explained by the prevention of enzyme leaching from Meso-Onion-S and the inhibition of enzyme denaturation due to multipoint linkages on the surface of LP molecules. Vesicle-like mesoporous silica was employed for the adsorption of Candida rugosa lipase.34 However, the enzyme stability experiment, performed with no incubation under rigorous shaking between enzyme activity measurements, resulted in a continuous decrease of enzyme activity. This result, together with the much better stability of NER-LP in Figure 4, suggests that the NER approach, via an addition of a simple step of enzyme cross-linking right after enzyme adsorption, is effective in preventing the leaching of enzyme and achieving an eventual success in a long-term stabilization of enzyme activity under rigorous shaking. Biodiesel Production by Immobilized Lipases in Meso-Onion-S. ADS-LP and NER-LP in Meso-Onion-S were used to catalyze the transesterification of soybean oil with methanol in isooctane for the production of biodiesels in the form of fatty acid esters. As shown in Figure 5, both

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CONCLUSIONS

Thermally stable mesoporous silica with highly ordered onionlike multilayer structure, denoted Meso-Onion-S, was synthesized by modifying the surfactant packing parameter with the same chemical composition for the synthesis of mesocellular siliceous foam (MCF) with a cell and window mesostructure. Meso-Onion-S was employed for the preparation of nanoscale enzyme reactors (NERs) by cross-linking adsorbed lipase, and highly curved pore structure of Meso-Onion-S was effective in improving the lipase stability by preventing the leaching of onedimensional cross-linked lipases from Meso-Onion-S. Stabilized lipase in the form of NERs was successfully employed for the transesterification of soybean oil with methanol in isooctane, which generated biodiesels in the form of fatty acid methyl esters. It is anticipated that the NER protocol using MesoOnion-S can be easily extended to the stabilization of various other enzymes, and the stabilized NERs can be employed in various enzyme applications including biodiesel production.

Figure 5. Time course of the transesterification reaction of soybean oil with methanol in isooctane using immobilized lipases. A reaction mixture of soybean oil/methanol (0.2 mmol/0.3 mmol) in 10 mL of isooctane was incubated under shaking (250 rpm) at 45 °C.

samples showed two phases in their conversion−time profiles, which can be explained by heterogeneous population in each sample; one is labile and the other is more stable. In the first 928

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ASSOCIATED CONTENT

S Supporting Information *

SEM image of Meso-Onion-S, TEM images of Meso-Onion-SX materials obtained at different hydrothermal reaction times (X), nitrogen adsorption−desorption isotherms and pore size distributions of Meso-Onion-S-X, and small angle X-ray scattering pattern of Meso-Onion-S-X (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax +82-54-279-5528, tel. 82-54-279-2395, e-mail jinwoo03@ postech.ac.kr (J.L.). Fax +82-2-886-8457, tel. 82-2-880-7150, email [email protected] (T.H.). Fax +82-2-926-6102, tel. 82-23290-4850, e-mail [email protected] (J.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Portions of this work were supported by grants from the National Research Foundation (NRF) funded by the Korean Ministry of Education, Science & Technology (MEST) (No. 2009-0082314, No. 2009-0059861, No. K2090300181211E0100-01710, and No. 2009-0084771) and by the Seoul R&BD Program (10920). T.H. would like to thank the financial support by the Korean Ministry of Science and Technology through the National Creative Research Initiative Program. J.L. was supported by the KRICT OASIS Project from Korea Research Institute of Chemical Technology.

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dx.doi.org/10.1021/cm202125q | Chem. Mater. 2012, 24, 924−929