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Comparative studies on catalytic properties of immobilized Candida rugosa lipase in ordered mesoporous rod-like silica and vesicle-like silica
Microporous and Mesoporous Materials 119 (2009) 223–229
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Comparative studies on catalytic properties of immobilized Candida rugosa lipase in ordered mesoporous rod-like silica and vesicle-like silica Guowei Zhou 1, Yijian Chen, Shihe Yang * Department of Chemistry, William Mong Institute of Nano Science and Technology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
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Article history: Received 29 February 2008 Received in revised form 17 September 2008 Accepted 16 October 2008 Available online 26 October 2008 Keywords: Mesoporous materials Rod-like silica Vesicle-like silica Candida rugosa lipase Immobilization
a b s t r a c t A lipase from Candida rugosa (CRL) has been immobilized in mesoporous rod-like silica and vesicle-like (or onion-like) silica supports by physical adsorption. The mesostructures and characteristics of the two mesoporous materials before and after adsorption of CRL were characterized by HRTEM, FESEM, SAXRD and N2 adsorption–desorption experiments. The pore sizes of the rod-like silica and the vesicle-like silicas were determined to be ca. 11 and 12 nm, respectively, larger than enough to accommodate lipase molecules (4 nm). The structural integrity of the rod-like silica as well as the vesicle-like silica is retained after adsorption of CRL into the mesopores. The catalytic activity, thermostability, and operational stability of the immobilized CRL were measured in phosphate buffer solution by hydrolysis of tributyrin. When the lipase immobilized in the vesicle-like silica was used as biocatalyst for hydrolysis of tributyrin in a batch process, the hydrolysis activity decreased only slightly from 74% to 60% over 10 cycles of reuse, indicating no obvious leaching out of the lipase from the round channels. The activity of vesicle-like silica immobilized CRL for catalytic hydrolysis of tributyrin is 21% and 14% higher than that of rod-like silica immobilized CRL at incubation temperature of 60 °C at 30 min and 90 min, respectively. The results demonstrate that the lipase immobilized in vesicle-like silica is superior to the rod-like silica in terms of higher activity, reusability and thermal stability. The unique curvature and the large size of the interlamellar pores of vesicle-like silica are suggested to be possible factors in achieving the higher activity and stability. Such a distinctive mesoporous geometry of the vesicle-like silica promises technological applications in protein adsorption and catalysis. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Immobilization of enzymes in inorganic matrices is very useful in practical applications due to the preserved stability and catalytic activity of the immobilized enzymes under extreme conditions. The past decade has witnessed a rapid progress in the synthesis of periodic mesoporous silicas (MPS) with uniform pore diameters of 2–30 nm and various morphologies such as fiber bundle, disklike, doughnut-like, rod-like, vesicle-like and helices using cationic, neutral, and block copolymer [1–9]. These materials are attractive for a range of applications in biotechnology, including purification, environmental management and enzymatic catalysis. In particular, the application of mesoporous materials in separation of proteins and enzyme immobilization is the most widely pursued. Pinnavaia et al. [8,9] first reported the feasibility of forming mesoporous vesicular silica with interlamellar pore of 2–2.7 nm by utilizing Bola-type surfactants H2N(CH2)nNH2 (n = 12–22) as structure directors in aqueous solution. They also reported the * Corresponding author. Tel.: +86 2358 7362. E-mail address: [email protected] (S. Yang). 1 Present address: School of Chemical Engineering, Shandong Institute of Light Industry, Jinan 250353, PR China. 1387-1811/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2008.10.013
preparation of ultrastable mesostructured silica vesicles with the interlamellar pore of 2.7–4.0 nm through supramolecular assembly of electrically neutral Gemini-type surfactants CnH2n + 1NH(CH2)2NH2 [10,11]. Subsequently, different types of amphiphiles were used as templates for the synthesis of vesicle-like silica [12–19]. However, the various routes for preparing vesicular silicas have so far resulted in small pores. Such small pores of the mesoporous vesicular silica may limit their application scope from adsorption and separation processes of large molecule proteins. Enzymes catalyzed reactions have high chemo-, regio-, and stereo-selectivity. However, un-supported enzymes are often denatured and frequently have limited stability in industrial or medical applications. Fortunately, immobilization of the enzymes onto a solid structure has been shown to be able to solve the problems [20–24]. As a result, it increases thermal and chemical stabilities of the enzymes, ensures their reusability, minimizes the cost of product isolation, provides operational flexibility, and thus enhance the productivity of biocatalytic processes [25–27]. Among the various enzyme immobilization schemes, adsorption, which involves different types of interactions between the carrier and the protein such as van der Waals interaction as well as hydrophobic and electrostatic interactions, has been used most frequently due
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to the extreme simplicity, easy product separation, reusability, and workability in unfriendly conditions. The first immobilized enzyme onto mesoporous silica MCM-41 was reported by Balkus et al. [20] in 1996. The immobilized globular enzymes included cytochrome c (bovine heart), papain (papaya latex) and trypsin (bovine pancreas). Since then, it has been established that many factors have a strong influence on enzyme adsorption and on the activity of the resultant biocatalyst, including the relative sizes of the mesopores and the enzyme, surface area, pore size distribution, mesopore volume, ionic strength, isoelectric point, pH value, and surface characteristics of both the support and the enzyme. The immobilization on mesoporous silica of different proteins and enzymes, such as cytochrome c [21,28], xylanase [22], horseradish peroxidase [25,26], subtilisin [29], chloroperoxidase [30], lysozyme [31–33], glucose oxidase [34,35], has been recently investigated. Among the various enzymes, lipases are of great interest owing to their unique properties and applications. Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are enzymes that catalyze the hydrolysis of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol, and are thus widely used in dairy industry, oil processing, production of surfactants, flavor and aroma compounds, and enantiomerically pure pharmaceuticals. A number of recent reports investigated immobilization of different lipases on mesoporous silica [27,36–40]. Li et al. [39] reported physical adsorption on SBA-15 of Newlase F, which contains lipase and acid protease from Rubus niveus, and they studied the enzymatic hydrolysis of protected dipeptide alkyl esters (nheptyl). Dumitriu et al. [37] immobilized the lipase from Candida Antarctica B (CALB) on MCM-36 by physical adsorption. The acylation of alcohols (1-butanol and 1-octanol) by vinyl esters (vinyl acetate and vinyl stearate) was used as a test reaction in order to evaluate the catalytic activity of the MCM-36 immobilized lipase. Duan et al. [38] achieved the first immobilization of the Porcine Pancreatic lipase (PPL) in the channels of MCM-41 supports, and then for preventing leach of the weakly bound lipase, the mouths of the channels were subsequently reduced by covalent coupling with an organic siloxane. Monduzzi and co-workers [36] investigated the physical and chemical adsorption of Mucor Javanicus lipase on SBA-15 mesoporous silica. Also, lipase from Candida Antarctica B (CALB) was successfully entrapped in the cagelike pores of siliceous mesocellular foam (MCF) using a pressure-driven method [27]. To our knowledge, however, there has been no report to date on the immobilization of lipases in vesicle-like silica. By introducing a hydrophobic additive TIPB in the hydrothermal synthesis of SBA-15, we are able to tune both the framework, from rod-like to vesicle-like, and the pore size, from 9 nm to 12 nm, of the porous silicas [41]. This paper first gives a brief account of the synthesis of the size-tunable rod-like and vesicle-like mesoporous silica by merely adjusting the molar ratio of TIPB:P123, and then mainly presents a comparative study of the mesoporous silica as carriers for the immobilization of a lipase from Candida rugosa (CRL). This is the first time the vesicle-like mesoporous silica is used as an enzyme-support for the immobilization of CRL, in fact any enzyme. The activity, reusability and thermal stability of immobilized CRL have been tested as biocatalyst for the hydrolysis of tributyrin. The results demonstrate that the properties of the lipase immobilized into the vesicle-like mesoporous silica are superior to that into the rod-like mesoporous silica. 2. Experimental 2.1. Chemicals Triblock copolymer Pluronic P123 (poly(ethylene oxide)-blockpoly(propylene oxide)-block-poly(ethylene oxide), EO20PO70EO20,
Mw = 5800), TEOS (tetraethoxysilane), lipase from Candida rugosa, tributyrin, potassium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Aldrich. TIPB (1,3,5-triisopropylbenzene) was purchased from Fluka. These chemicals were used as received without further purification. Millipore water 18.3 M X cm (purified using an EASYpure RF ultrapure water system) was used in all of the experiments. 2.2. Synthesis of mesoporous rod-like and vesicle-like silica A typical preparation procedure is given here. First, 1.0 g of P123 was dissolved in a mixture of 8.5 g of H2O and 30 g of 2 M HCl aqueous solution and the resulting solution was stirred at room temperature until the solution became clear. Into the solution specified amounts of TIPB (with the molar ratio of TIPB:P123 being 2.9:1 and 23.2:1, respectively) were added drop-wise for the preparation of a set of samples. The mixture was stirred at room temperature for another 18 h. Second, 2.1 g of TEOS was added into this solution under stirring for 8 min and the mixture was kept under static conditions at 35 °C for 24 h. This mixture solution was then transferred into a teflon-lined autoclave and heated to and kept at 130 °C for 24 h. Finally, white precipitates were filtered, washed with water, air-dried at room temperature, and calcined at 500 °C for 6 h in a tube furnace to remove the organic templates. 2.3. Characterization High resolution transmission electron microscopy (HRTEM) was performed using a JEOL 2010 electron microscope. Samples for HRTEM measurements were prepared by dipping a carbon-coated copper grid into a suspension of ground samples in ethanol, which were pre-sonicated for 20 min. Field emission scanning electron microscope (FESEM) measurements were carried out with a JEOL JSM-6700F microscope. Samples were deposited on the surface of silicon wafer by dropping a suspension of ground samples in ethanol that was pre-sonicated for 20 min, and sputter coated for 2 cycles with gold. The accelerating voltage used was 5 kV with an electric current of 10 lA. Nitrogen adsorption–desorption isotherms were measured at 196 °C on a SA3100 surface area and pore size analyzer. Samples were degassed in a vacuum at 200 °C for 3 h prior to each measurement, while the lipase loaded samples were outgassed at 35 °C for 12 h. Brunauer-Emmett-Teller (BET) method was utilized to calculate the specific surface areas (SBET) from the adsorption data. Pore size distributions were derived from the adsorption branches of isotherms by using the Barrett-JoynerHalenda (BJH) model. Small-angle X-ray diffraction (SAXRD) patterns were recorded on a X’pert Pro powder X-ray diffractometer with Cu Ka radiation (40 kV, 40 mA), in reflection mode (2h = 0.5–5°) with a step size of 0.02° and a scan step time of 5 s. 2.4. Adsorption of lipase onto mesoporous supports Five milliliter of 50 mM sodium phosphate buffer (pH 6.0) were added to 40 mg mesoporous silica support in 20 mL capped vials. After the mixture was sonicated for 20 min, 5 mL of lipase stock solution with a concentration of 8 mg mL 1 in 50 mM phosphate buffer at pH 6.0 was added. The mixture was stirred by a magnetic stirrer at room temperature for different time periods (1–48 h). The supernatant was separated from the solid material by centrifugation (12000 rpm, 8 min, 4 °C). The amount of lipase immobilized onto the rod-like or vesicle-like silica was assayed by subtracting the amount in the supernatant liquid after adsorption from the amount of CRL present before addition of the mesoporous silicas, determined by UV–vis spectrophotometry at 258 nm; the supernatant liquid was isolated by centrifugation before the UV–vis mea-
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surements. The immobilized lipase was washed with phosphate buffer (pH 6.0, 10 mL) solution, dried overnight under vacuum at room temperature and then stored at 4 °C in a refrigerator. 2.5. Activity assays Activity of the immobilized lipase (with the lipase loading of 408 and 363 mg g 1 onto the rod-like and vesicle-like silica, respectively, after 16 h of adsorption) was determined by measuring the hydrolysis of tributyrin by the pH-stat method using a 310 Thermo Orion Perphect Meter [36]. In the hydrolysis reaction of tributyrin, butyric acid was produced as a by-product, which lowered pH of the hydrolysis mixture. The activity of the immobilized enzyme could then be determined by titrating the butyric acid with NaOH so as to maintain a constant pH. From the consumption of NaOH, the amount of butyric acid produced was obtained. The activity of the immobilized lipase was expressed as the percentage conversion of the tributyrin. Experiments with free lipase were performed by adding a desired amount of lipase. 2.6. Assay procedure A mixture of tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer saline (5 mL) was vigorously stirred at room temperature for 30 min, forming a tributyrin emulsion [38]. After pH of the emulsion was stabilized, the immobilized lipase (16 mg CRL) was added to the emulsion. The mixture was stirred for 6 h, and then centrifuged (12000 rpm, 8 min, 4 °C). The supernatant was titrated with 0.1 M sodium hydroxide solution so as to maintain a constant pH value. The volume of sodium hydroxide consumed was recorded and the activity of the immobilized CRL calculated. The activity of the free lipase (16 mg) was assayed by the same method. The same amounts (40 mg) of mesoporous silica were also used to measure the catalytic activity as blank experiments. Blank results showed that neither the mesoporous silicas nor the substrate consumed significant amounts of NaOH solution, indicating that assaying the activity of immobilized lipase by titration with NaOH is a viable procedure. 2.7. Thermal stability and operational stability Thermal stability experiments were first conducted by heating a solution of free lipase (16 mg CRL was solved in 2.5 mL phosphate buffer) or a suspension of immobilized lipase (immobilized lipase was dispersed in 2.5 mL phosphate buffer) in capped vials in a water bath at a desired temperature (23, 50, 60, 70 oC) for 30 min or at 60 oC for a desired amount of time (30, 60, 90 min). The solutions were allowed to cool down to room temperature. Afterwards, the mixture emulsion of tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (2.5 mL) was added for the hydrolysis of tributyrin. The operational stability of immobilized lipase was tested by repeating batch experiments (10 times) using the same substrate concentration, temperature and the reaction time as described above. 3. Results and discussion 3.1. Structures of the mesoporous rod-like and vesicle-like silicas Representative HRTEM images in Fig. 1 show clearly the rodlike and vesicle-like morphologies of the mesoporous silicas we obtained. Without TIPB, SBA-15 silica with a rod-like morphology of uniform size has been obtained similar to that prepared by Sayari et al. [3]. When a small amount of TIPB was added (TIPB to P123 molar ratio = 2.9:1), the morphology of the particles remained
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rod-like with hexagonal channels (Fig. 1a and b), but the pore diameter was increased from 9 nm to 11 nm. However when a large amount of TIPB was added, an unexpected drastic change occurred in the morphology from rod-like silica to vesicle-like or onion-like silica as shown in Fig. 1c and d. Almost all of the particles can be described as being multilamellar vesicles with a framework structure made of undulated silica layers ca. 5 nm thick with regularly spaced mesopores (12 nm); such an onion structure extends from the outer surface all the way inward to the center of the vesicle. As can be seen from the typical FESEM image presented in Fig. 2, many vesicle-like silica particles are ruptured obviously due to the calcination step, whereby gases inside the vesicles bustle out at the high temperatures. These breaches (20 nm) of vesicle-like silica can afford the entrance for the enzyme adsorption. 3.2. Lipase immobilization into mesoporous rod-like silica and vesiclelike silica The isoelectric point (pI or the pH at which the material has an equal number of positive and negative charges at its surface) of an enzyme is often considered for adsorption studies. The pI of CRL is 5.2 and hence, the protein is positively charged at a pH below pI and negatively charged at a pH above pI. On the other hand, the pI of the silica surface of SBA-15 support is about 3.7 ± 0.3 [22], Possible physical adsorption forces of CRL on mesoporous silica involved here include weak van der Waals interaction, hydrogen bonding interaction, and electrostatic interactions between the amino acid residues on the surface of CRL and silanol groups on the surface of the mesoporous materials [23], with the last being of the highest magnitude. When the solution pH is higher than the pI of CRL, the surface of the protein becomes negatively charged and therefore an electrostatic repulsion between the negatively charged CRL and the negatively charged silanol groups mesoporous silica support reduces the amount of CRL adsorption. At a pH near the isoelectric point, the net charge of the lipase is zero and hence the electrostatic interaction between the protein molecules is the lowest. The diminished electrostatic repulsion between the CRL molecules allows them to pack closer together onto the surface of the adsorbent in physical adsorption [23]. Proteins are known to show maximal adsorption when solution pH is near pI of protein [36], so we selected the solution with pH 6.0, which is near the pI of CRL. The lipase can enter the rod-like silica channels from their opened ends, while it can enter the vesicle-like silica curved channels from the breaches formed during calcination. The mesoporous rod-like and vesicle-like silicas were characterized by SAXRD and nitrogen adsorption before and after protein adsorption. The SAXRD patterns of the rod-like and the vesicle-like silicas before and after adsorption at an initial CRL concentration of 4 mg mL 1 at pH 6.0 are shown in Figs. 3 and 4, respectively. The rod-like silica before adsorption exhibits a strong reflection peak (1 0 0) and three small peaks (1 1 0, 2 0 0, 2 1 0) (Fig. 3a), characteristic of the hexagonal mesoporous structure. After lipase adsorption (Fig. 3b), the low angle reflection peak (1 0 0) can also be obtained, but the higher angle reflection peaks (1 1 0 and 2 0 0) are not observed, clearly reflecting the filling of the channels with the enzyme molecules. Similarly, the SAXRD patterns of the vesicle-like silica before (Fig. 4a) and after (Fig. 4b) adsorption of CRL show at least two well-resolved diffraction peaks (0 0 1 and 0 0 2) typical of the lamellar framework structure, but again, the higher-order reflection peak has suffered a significant intensity loss due to the enzyme adsorption. The fact that all of the original diffraction peaks are observed after enzyme adsorption indicates that both the rod-like and the vesicle-like silica maintain their longrange framework orders after the lipase loading [21,42]. Therefore we can conclude that the lipase molecules can be packed inside the
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Fig. 1. Representative HRTEM images of rod-like silica (a, b) and vesicle-like silica (c, d).
Fig. 3. SAXRD patterns of the rod-like silica before (a) and after (b) adsorption of CRL in pH 6.0 phosphate buffer solution for 16 h at an initial concentration of 4 mg mL 1 (CRL). Fig. 2. Representative FESEM image of vesicle-like silicas. The arrows point to the ruptured vesicle-like silicas.
mesopores without significantly affecting the structural integrity of the rod-like and vesicle-like silicas. The nitrogen adsorption–desorption isotherms of the rod-like and the vesicle-like silica and the resulting Barrett-Joyner-Halenda (BJH) pore size distributions determined from the corresponding adsorption branches before and after loading of CRL are shown in Figs. 5 and 6, respectively. One can see that the amount of nitrogen
adsorbed decreases markedly in the rod-like silica (from 687 cm3 g 1 to 472 cm3 g 1) and the vesicle-like silica (from 1028 cm3 g 1 to 703 cm3 g 1) upon CRL adsorption. Similar results have been reported for the adsorption of lysozyme and cytochrome c onto MCM-41 and SBA-15 [21,33]. The nitrogen adsorption– desorption isotherms of the rod-like silica before and after adsorption of CRL are of type IV with H1-type hysteresis loop that is characteristic of SBA-15. In contrast, the sorption isotherms of vesiclelike silica before and after adsorption of CRL are different from a typical type IV of SBA-15, e.g., they have a broad hysteresis loop
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Fig. 4. SAXRD patterns of vesicle-like silica before (a) and after (b) adsorption of CRL in pH 6.0 phosphate buffer solution for 16 h at an initial concentration of 4 mg mL 1 (CRL).
Fig. 6. N2 adsorption–desorption isotherms (a) and pore diameter distribution curves (b) of vesicle-like silica before (j) and after (d) lipase loading, and after lipase loading as well as after use for 10 runs of lipase catalysed hydrolysis of tributyrin (N).
Table 1 Structural parameters of the rod-like and vesicle-like silicas. TIPB:P123 molar ratio
SBET (m2 g
0 2.9/1 23.2/1
459 437 518
*
Fig. 5. N2 adsorption–desorption isotherms (a) and pore diameter distribution curves (b) of rod-like silica before (d) and after (j) lipase loading.
in the range of P/P0 = 0.51–0.99, which does not close until the saturation pressure is reached (Fig. 6a). Similar results were reported for the vesicle-like silica prepared by Lind et al. [13]. This result may be attributed to N2 filling the multilamellar mesopores of vesicular silica [10,13]. The vesicle-like silica before and after loading of lipase exhibit pore size distribution maxima centered at 12.3 and 11.1 nm, respectively (Fig. 6b). After 10 times of use for catalytic hydrolysis reactions, the vesicle-like silica immobilized lipase can maintain the shape of the isotherms of the original vesicle-like silica (Fig. 6a) and show pore size distribution maximum still centered at 11.1 nm (Fig. 6b). Moreover, the vesicle-like silica exhibits a much larger Brunauer-Emmett-Teller (BET) surface area SBET (520 m2 g 1) than that of the rod-like silica (440 m2 g 1) as well as a much larger total adsorption pore volume of 1.61 cm3 g 1 at a relative pressure of 0.99 (Table 1). Table 2 summarizes the textural properties of the rod-like and vesicle-like silicas after CRL loading and the data of catalytic reac-
Vp* (m3 g
1
)
1
)
0.95 1.09 1.61
WBJH (nm)
Morphology
d100spacing (nm)
9.2 11.1 12.3
Rod-like Rod-like Vesicle-like
9.7 10.6
d001spacing (nm)
11.8
The pore volume was obtained at relative pressure of 0.99.
tions for CRL immobilized in the vesicle-like silica. By comparing Table 2 and Table 1, one can see that the specific surface areas and the total pore volumes of the rod-like and vesicle-like silicas were drastically reduced after CRL adsorption. The specific surface area and the total pore volume of the rod-like silica decreased from 437 to 263 m2 g 1 and from 1.09 to 0.76 cm3 g 1, respectively, upon adsorption of CRL. More significantly, the adsorption of CRL led to the decrease of the specific surface area and the total pore volume of the vesicle-like silica from 518 to 235 m2 g 1 and from 1.61 to 1.11 cm3 g 1, respectively. The observed decrease in pore
Table 2 Structural parameters of the rod-like and vesicle-like silicas after CRL adsorption and after different numbers of runs for catalytic hydrolytic reactions. TIPB:P123 molar ratio
Number of reaction runs
SBET (m2 g
2.9:1 23.2:1 23.2:1 23.2:1
0 0 1 10
249 235 247 187
1
)
Vp (cm3 g 0.70 1.11 1.03 0.90
1
)
WBJH (nm) 11.1 12.3 11.1 11.1
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volume of 0.33 cm3 g 1 and specific surface area of 174 m2 g 1 for rod-like silica, and in pore volume of 0.50 cm3 g 1 and specific surface area of 283 m2 g 1 for vesicle-like silica after loading with CRL can be mainly attributed to the adsorption of CRL into the mesopores. However, other scenarios also need be considered. First, rod-like and vesicle-like silicas possess a large quantity of (ultra)micropores, therefore, some of the CRL molecules can block the pore mouths of the (ultra)micropores present in the silica walls. So, these micropores can no longer adsorb nitrogen, resulting in a reduction of the pore volume and specific surface area after CRL adsorption. Second, the increasing weight of rod-like and vesicle-like silicas after adsorption of CRL can also reduce the pore volume and specific surface area. Third, the tight packing of CRL molecules instead of adsorption in the mesopores may also decrease the pore volume and specific surface area. After 1 and 10 runs of catalytic hydrolytic reactions, the specific surface area and the total pore volume of the vesicle-like silica were reduced to 247 and 187 m2 g 1 and to 1.03 and 0.90 cm3 g 1, respectively. A possible explanation for this is a partial loss of the structural order of the vesicle-like silica after the 10 runs of the catalytic reactions. 3.3. Adsorption kinetics The adsorption kinetic curves of CRL into rod-like silica and vesicle-like silica in pH 6.0 solution at room temperature are shown in Fig. 7. We can find that the adsorption rate of CRL into rod-like silica is faster (reaching 193 mg g 1 within 1 h at room temperature) than that of vesicle-like silica (reaching 95 mg g 1 within 1 h at room temperature) and the amount of CRL immobilized into rodlike silica is higher (reaching 506 mg g 1 for 48 h at room temperature) than that of vesicle-like silica (reaching 476 mg g 1 for 48 h at room temperature). The significant difference in the adsorption rate and the adsorbed amount of CRL into rod-like silica and vesicle-like silica can be reasonably attributed to their different morphologies [31]. First, for rod-like silicas, there are at least two open entrances in the ends of each rod for lipase adsorption, whereas in the vesicle-like silicas, the entrances are limited to the breaches formed during calcination. Second, the mesochannel of rod-like silica is straight, whereas the mesochannel of vesiclelike silica is curved, which may influence the transport of the CRL. Therefore, the mesoporous morphologies appear to play an important role in their immobilization performance of enzyme. 3.4. Activity and stability of immobilized lipase
Our data show that the initial activity (the percentage conversion of tributyrin in the first run, 74%) of immobilized CRL on the vesicle-like silica is higher than that (66%) on the rod-like silica (Fig. 8). Moreover, after 10 successive batches of catalytic reactions, the hydrolysis activity of immobilized CRL on the vesicle-like silica reduced from 74% to 60%, whereas only 51% of the hydrolysis activity is retained for that on the rod-like silica. The decreased activities for the two immobilized CRL samples are mainly caused by lipase leakage owing to the multiple soaking and separation processes during the successive hydrolysis reactions. The better initial activity and operational stability of the vesicle-like silica immobilized CRL than the rod-like silica immobilized CRL is probably due to the unique flexual structure and the larger size of the mesochannels in the vesicle-like silica as well as the added inter-shell flexibility arising from the special vesicular structure. These factors would increase the affinity interaction between the silanol groups of vesicle-like silica and the lipase, resulting in a slower lipase leakage during the recycling process. The storage stability of immobilized CRL was also evaluated by running the catalytic reactions one time per week for 10 weeks (data not shown). The results are similar to those presented above, for immobilized CRL in both the rod-like and the vesicle-like silicas, demonstrating the good long-term storage stability of the immobilized lipase. The thermal stability of free and immobilized CRL was investigated and compared for catalytic hydrolysis of tributyrin by incubating the lipase for 30 min at different temperatures (Fig. 9) or
Fig. 8. Conversion of tributyrin versus the reaction run numbers. (j) Rod-like silica immobilized CRL; (d) Vesicle-like silica immobilized CRL. Hydrolysis conditions: tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (5 mL).
As mentioned above, one of the advantages of the immobilized enzymes is their reusability without compromising the activity.
Fig. 7. Adsorption kinetic curves of CRL into rod-like silica (j) and vesicle-like silica (d) in pH 6.0 solution at room temperature.
Fig. 9. Effect of thermal incubation temperature on conversion of tributyrin (incubation time: 30 min). (j) Rod-like silica immobilized CRL; (N) Free CRL; and (d) Vesicle-like silica immobilized CRL. Hydrolysis conditions: tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (5 mL).
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curvature of the mesochannels in the vesicle-like silica as well as the added inter-shell flexibility arising from the large size of the pores. Our study is the first to reveal the superior properties of the novel vesicle-like silica in protein adsorption and enzymatic catalysis and paves the way to the technological applications of this class of interesting materials. Acknowledgment We acknowledge supports from the Hong Kong University of Science and Technology (RPC06/07.SC03) and NNSFC-RGC (N_HKUST604/04) and RGC (604206) administrated by the UGC of Hong Kong. Fig. 10. Effect of incubation time on conversion of tributyrin (incubation temperature 60 °C). (j) Rod-like silica immobilized CRL; (N) Free CRL; and (d) Vesicle-like silica immobilized CRL. Hydrolysis conditions: tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (5 mL).
incubating the lipase at 60 °C for different times (Fig. 10). The catalytic activity of the vesicle-like silica immobilized CRL for hydrolysis of tributyrin is 67% and 46% when incubated at temperature of 60 °C for 30 min and 90 min, respectively, whereas it is merely 46% and 32% with the rod-like silica immobilized CRL at the same condition. It can also be seen that the vesicle-like silica immobilized CRL shows a higher thermal stability than the free CRL. In contrast, the rod-like silica immobilized CRL exhibits a reduced hydrolysis activity after the thermal treatment relative to that of the free lipase. Similarly, Hudson et al. [22] reported that cytochrome c immobilized onto SBA-15 showed a higher intrinsic activity but lower thermostability than free cytochrome c. We can conclude that the CRL immobilized onto the vesicle-like silica shows resistance to leaching and an enhanced activity compared to the rod-like silica immobilized CRL. A likely scenario is that the active sites of the immobilized lipase into the rod-like silica are more susceptible to heat denaturation. The higher stability and activity of the lipase immobilized in vesicle-like silicas makes them an excellent candidate for a new bioimmobilization host. 4. Conclusions The vesicle-like silica we have developed possesses large uniform interlamellar pores and a completely multilamellar structure in the whole vesicular spheres. These features are advantageous for applications in many technological areas involving adsorption of large molecules such as proteins and enzyme catalysis. For comparative studies, lipase from Candida rugosa (CRL) has been immobilized in the rod-like and vesicle-like silicas by physical adsorption. The SAXRD and N2 adsorption–desorption data after CRL adsorption reveal that the mesostructure can be maintained and the CRL molecules are efficiently adsorbed inside the mesopores of the rod-like silica and the vesicle-like silicas. The catalytic activity, thermostability, and operational stability of the immobilized lipase were measured through the hydrolysis of tributyrin in phosphate buffer solution. After 10 runs of catalytic hydrolysis of tributyrin, the catalytic hydrolysis activity of immobilized CRL on the vesicle-like silica is reduced from 74% to 60%, whereas more reduction from 66% to 51% is found for the immobilized CRL on the rod-like silica. When incubated at temperature of 60 °C for 30 min and 90 min, the catalytic activity of the vesicle-like silica immobilized CRL for hydrolysis of tributyrin is, respectively, 67% and 46%, whereas it is merely 46% and 32% with the rod-like silica immobilized CRL. The lipase immobilized into the vesicle-like silica shows substantially higher activity, reusability and thermal stability compared to that into the rod-like silica due probably to the unique
References [1] D. Zhao, Q. Huo, J. Feng, B.F. Chmelka, G.D. Stucky, J. Am. Chem. Soc. 120 (1998) 6024. [2] D. Zhao, J. Feng, Q. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka, G.D. Stucky, Science 279 (1998) 548. [3] A. Sayari, B.-H. Han, Y. Yang, J. Am. Chem. Soc. 126 (2004) 14348. [4] J.D. Rimer, J.M. Fedeyko, D.G. Vlachos, R.F. Lobo, Chem. Eur. J. 12 (2006) 2926. [5] S. Che, K. Lund, T. Tatsumi, S. Iijima, S.H. Joo, R. Ryoo, O. Terasaki, Angew. Chem. Int. Ed. 42 (2003) 2182. [6] N.E. Botterhuis, Q. Sun, P.C.M.M. Magusin, R.A.V. Santen, N.A.J.M. Sommerdijk, Chem. Eur. J. 12 (2006) 1448. [7] F. Hoffmann, M. Cornelius, J. Morell, M. Froba, Angew. Chem. Int. Ed. 45 (2006) 3216. [8] P.T. Tanev, T.J. Pinnavaia, Science 271 (1996) 1267. [9] P.T. Tanev, Y. Liang, T.J. Pinnavaia, J. Am. Chem. Soc. 119 (1997) 8616. [10] S.S. Kim, W. Zhang, T.J. Pinnavaia, Science 282 (1998) 1302. [11] S.S. Kim, Y. Liu, T.J. Pinnavaia, Micropor. Mesopor. Mater. 44–45 (2001) 489. [12] A.J. Karkamkar, S.S. Kim, S.D. Mahanti, T.J. Pinnavaia, Adv. Funct. Mater. 14 (2004) 507. [13] A. Lind, B. Spliethoff, M. Linden, Chem. Mater. 15 (2003) 813. [14] H.-P. Hentze, S.R. Raghavan, C.A. McKelvey, E.W. Kaler, Langmuir 19 (2003) 1069. [15] R.K. Rana, Y. Mastai, A. Gedanken, Adv. Mater. 14 (2002) 1414. [16] J.G.C. Shen, J. Phys. Chem. B 108 (2004) 44. [17] J. Sun, D. Ma, H. Zhang, C. Wang, X. Bao, D.S. Su, A. Klein-Hoffmann, G. Weinberg, S. Mann, J. Mater. Chem. 16 (2006) 1507. [18] Y.Q. Yeh, B.C. Chen, H.P. Lin, C.Y. Tang, Langmuir 22 (2006) 6. [19] J. Liu, Q. Yang, L. Zhang, D. Jiang, X. Shi, J. Yang, H. Zhong, C. Li, Adv. Funct. Mater. 17 (2007) 569. [20] J.F. Diaz, K.J. Balkus, J. Mol. Catal. B 2 (1996) 115. [21] A. Vinu, V. Murugesan, O. Tangermann, M. Hartmann, Chem. Mater. 16 (2004) 3056. [22] S. Hudson, E. Magner, J. Cooney, B.K. Hodnett, J. Phys. Chem. B 109 (2005) 19496. [23] H.H.P. Yiu, P.A. Wright, J. Mater. Chem. 15 (2005) 3690. [24] D. Moelans, P. Cool, J. Baeyens, E.F. Vansant, Catal. Commun. 6 (2005) 307. [25] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Micropor. Mesopor. Mater. 44–45 (2001) 755. [26] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Chem. Mater. 12 (2000) 3301. [27] Y. Han, S.S. Lee, J.Y. Ying, Chem. Mater. 18 (2006) 643. [28] Y. Wang, F. Caruso, Chem. Mater. 17 (2005) 953. [29] J. Deere, E. Magner, J.G. Wall, B.K. Hodnett, J. Phys. Chem. B 106 (2002) 7340. [30] J. Aburto, M. Ayala, I. Bustos-Jaimes, C. Montiel, E. Terres, J.M. Dominguez, E. Torres, Micropor. Mesopor. Mater. 83 (2005) 193. [31] J. Lei, J. Fan, C. Yu, L. Zhang, S. Jiang, B. Tu, D. Zhao, Micropor. Mesopor. Mater. 73 (2004) 121. [32] A. Katiyar, L. Ji, P. Smirniotis, N.G. Pinto, J. Chromatogr. A 1069 (2005) 119. [33] A. Vinu, V. Murugesan, M. Hartmann, J. Phys. Chem. B 108 (2004) 7323. [34] J.L. Blin, C. Cerardin, C. Carteret, L. Rodehuser, C. Selve, M.J. Stebe, Chem. Mater. 17 (2005) 1479. [35] K. Han, Z. Wu, J. Lee, I.S. Ahn, J.W. Park, B.R. Min, K. Lee, Biochem. Eng. J. 22 (2005) 161. [36] A. Salis, D. Meloni, S. Ligas, M.F. Casula, M. Monduzzi, V. Solinas, E. Dumitriu, Langmuir 21 (2005) 5511. [37] E. Dumitriu, F. Secundo, J. Patarin, I. Fechete, J. Mol. Catal. B: Enzymatic 22 (2003) 119. [38] H. Ma, J. He, D.G. Evans, X. Duan, J. Mol. Catal. B: Enzymatic 30 (2004) 209. [39] Z.Z. Chen, Y.M. Li, X. Peng, F.R. Huang, Y.F. Zhao, J. Mol. Catal. B: Enzymatic 18 (2002) 243. [40] M. Mureseanu, A. Galarneau, G. Renard, F. Fajula, Langmuir 21 (2005) 4648. [41] G. Zhou, Y. Chen, J. Yang, S. Yang, J. Mater. Chem. 17 (2007) 2839. [42] W. Hammond, E. Prouzet, S.D. Mahanti, T.J. Pinnavaia, Micropor. Mesopor. Mater. 27 (1999) 19.
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Comparative studies on catalytic properties of immobilized Candida rugosa lipase in ordered mesoporous rod-like silica and vesicle-like silica Guowei Zhou 1, Yijian Chen, Shihe Yang * Department of Chemistry, William Mong Institute of Nano Science and Technology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
a r t i c l e
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Article history: Received 29 February 2008 Received in revised form 17 September 2008 Accepted 16 October 2008 Available online 26 October 2008 Keywords: Mesoporous materials Rod-like silica Vesicle-like silica Candida rugosa lipase Immobilization
a b s t r a c t A lipase from Candida rugosa (CRL) has been immobilized in mesoporous rod-like silica and vesicle-like (or onion-like) silica supports by physical adsorption. The mesostructures and characteristics of the two mesoporous materials before and after adsorption of CRL were characterized by HRTEM, FESEM, SAXRD and N2 adsorption–desorption experiments. The pore sizes of the rod-like silica and the vesicle-like silicas were determined to be ca. 11 and 12 nm, respectively, larger than enough to accommodate lipase molecules (4 nm). The structural integrity of the rod-like silica as well as the vesicle-like silica is retained after adsorption of CRL into the mesopores. The catalytic activity, thermostability, and operational stability of the immobilized CRL were measured in phosphate buffer solution by hydrolysis of tributyrin. When the lipase immobilized in the vesicle-like silica was used as biocatalyst for hydrolysis of tributyrin in a batch process, the hydrolysis activity decreased only slightly from 74% to 60% over 10 cycles of reuse, indicating no obvious leaching out of the lipase from the round channels. The activity of vesicle-like silica immobilized CRL for catalytic hydrolysis of tributyrin is 21% and 14% higher than that of rod-like silica immobilized CRL at incubation temperature of 60 °C at 30 min and 90 min, respectively. The results demonstrate that the lipase immobilized in vesicle-like silica is superior to the rod-like silica in terms of higher activity, reusability and thermal stability. The unique curvature and the large size of the interlamellar pores of vesicle-like silica are suggested to be possible factors in achieving the higher activity and stability. Such a distinctive mesoporous geometry of the vesicle-like silica promises technological applications in protein adsorption and catalysis. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Immobilization of enzymes in inorganic matrices is very useful in practical applications due to the preserved stability and catalytic activity of the immobilized enzymes under extreme conditions. The past decade has witnessed a rapid progress in the synthesis of periodic mesoporous silicas (MPS) with uniform pore diameters of 2–30 nm and various morphologies such as fiber bundle, disklike, doughnut-like, rod-like, vesicle-like and helices using cationic, neutral, and block copolymer [1–9]. These materials are attractive for a range of applications in biotechnology, including purification, environmental management and enzymatic catalysis. In particular, the application of mesoporous materials in separation of proteins and enzyme immobilization is the most widely pursued. Pinnavaia et al. [8,9] first reported the feasibility of forming mesoporous vesicular silica with interlamellar pore of 2–2.7 nm by utilizing Bola-type surfactants H2N(CH2)nNH2 (n = 12–22) as structure directors in aqueous solution. They also reported the * Corresponding author. Tel.: +86 2358 7362. E-mail address: [email protected] (S. Yang). 1 Present address: School of Chemical Engineering, Shandong Institute of Light Industry, Jinan 250353, PR China. 1387-1811/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2008.10.013
preparation of ultrastable mesostructured silica vesicles with the interlamellar pore of 2.7–4.0 nm through supramolecular assembly of electrically neutral Gemini-type surfactants CnH2n + 1NH(CH2)2NH2 [10,11]. Subsequently, different types of amphiphiles were used as templates for the synthesis of vesicle-like silica [12–19]. However, the various routes for preparing vesicular silicas have so far resulted in small pores. Such small pores of the mesoporous vesicular silica may limit their application scope from adsorption and separation processes of large molecule proteins. Enzymes catalyzed reactions have high chemo-, regio-, and stereo-selectivity. However, un-supported enzymes are often denatured and frequently have limited stability in industrial or medical applications. Fortunately, immobilization of the enzymes onto a solid structure has been shown to be able to solve the problems [20–24]. As a result, it increases thermal and chemical stabilities of the enzymes, ensures their reusability, minimizes the cost of product isolation, provides operational flexibility, and thus enhance the productivity of biocatalytic processes [25–27]. Among the various enzyme immobilization schemes, adsorption, which involves different types of interactions between the carrier and the protein such as van der Waals interaction as well as hydrophobic and electrostatic interactions, has been used most frequently due
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to the extreme simplicity, easy product separation, reusability, and workability in unfriendly conditions. The first immobilized enzyme onto mesoporous silica MCM-41 was reported by Balkus et al. [20] in 1996. The immobilized globular enzymes included cytochrome c (bovine heart), papain (papaya latex) and trypsin (bovine pancreas). Since then, it has been established that many factors have a strong influence on enzyme adsorption and on the activity of the resultant biocatalyst, including the relative sizes of the mesopores and the enzyme, surface area, pore size distribution, mesopore volume, ionic strength, isoelectric point, pH value, and surface characteristics of both the support and the enzyme. The immobilization on mesoporous silica of different proteins and enzymes, such as cytochrome c [21,28], xylanase [22], horseradish peroxidase [25,26], subtilisin [29], chloroperoxidase [30], lysozyme [31–33], glucose oxidase [34,35], has been recently investigated. Among the various enzymes, lipases are of great interest owing to their unique properties and applications. Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are enzymes that catalyze the hydrolysis of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol, and are thus widely used in dairy industry, oil processing, production of surfactants, flavor and aroma compounds, and enantiomerically pure pharmaceuticals. A number of recent reports investigated immobilization of different lipases on mesoporous silica [27,36–40]. Li et al. [39] reported physical adsorption on SBA-15 of Newlase F, which contains lipase and acid protease from Rubus niveus, and they studied the enzymatic hydrolysis of protected dipeptide alkyl esters (nheptyl). Dumitriu et al. [37] immobilized the lipase from Candida Antarctica B (CALB) on MCM-36 by physical adsorption. The acylation of alcohols (1-butanol and 1-octanol) by vinyl esters (vinyl acetate and vinyl stearate) was used as a test reaction in order to evaluate the catalytic activity of the MCM-36 immobilized lipase. Duan et al. [38] achieved the first immobilization of the Porcine Pancreatic lipase (PPL) in the channels of MCM-41 supports, and then for preventing leach of the weakly bound lipase, the mouths of the channels were subsequently reduced by covalent coupling with an organic siloxane. Monduzzi and co-workers [36] investigated the physical and chemical adsorption of Mucor Javanicus lipase on SBA-15 mesoporous silica. Also, lipase from Candida Antarctica B (CALB) was successfully entrapped in the cagelike pores of siliceous mesocellular foam (MCF) using a pressure-driven method [27]. To our knowledge, however, there has been no report to date on the immobilization of lipases in vesicle-like silica. By introducing a hydrophobic additive TIPB in the hydrothermal synthesis of SBA-15, we are able to tune both the framework, from rod-like to vesicle-like, and the pore size, from 9 nm to 12 nm, of the porous silicas [41]. This paper first gives a brief account of the synthesis of the size-tunable rod-like and vesicle-like mesoporous silica by merely adjusting the molar ratio of TIPB:P123, and then mainly presents a comparative study of the mesoporous silica as carriers for the immobilization of a lipase from Candida rugosa (CRL). This is the first time the vesicle-like mesoporous silica is used as an enzyme-support for the immobilization of CRL, in fact any enzyme. The activity, reusability and thermal stability of immobilized CRL have been tested as biocatalyst for the hydrolysis of tributyrin. The results demonstrate that the properties of the lipase immobilized into the vesicle-like mesoporous silica are superior to that into the rod-like mesoporous silica. 2. Experimental 2.1. Chemicals Triblock copolymer Pluronic P123 (poly(ethylene oxide)-blockpoly(propylene oxide)-block-poly(ethylene oxide), EO20PO70EO20,
Mw = 5800), TEOS (tetraethoxysilane), lipase from Candida rugosa, tributyrin, potassium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Aldrich. TIPB (1,3,5-triisopropylbenzene) was purchased from Fluka. These chemicals were used as received without further purification. Millipore water 18.3 M X cm (purified using an EASYpure RF ultrapure water system) was used in all of the experiments. 2.2. Synthesis of mesoporous rod-like and vesicle-like silica A typical preparation procedure is given here. First, 1.0 g of P123 was dissolved in a mixture of 8.5 g of H2O and 30 g of 2 M HCl aqueous solution and the resulting solution was stirred at room temperature until the solution became clear. Into the solution specified amounts of TIPB (with the molar ratio of TIPB:P123 being 2.9:1 and 23.2:1, respectively) were added drop-wise for the preparation of a set of samples. The mixture was stirred at room temperature for another 18 h. Second, 2.1 g of TEOS was added into this solution under stirring for 8 min and the mixture was kept under static conditions at 35 °C for 24 h. This mixture solution was then transferred into a teflon-lined autoclave and heated to and kept at 130 °C for 24 h. Finally, white precipitates were filtered, washed with water, air-dried at room temperature, and calcined at 500 °C for 6 h in a tube furnace to remove the organic templates. 2.3. Characterization High resolution transmission electron microscopy (HRTEM) was performed using a JEOL 2010 electron microscope. Samples for HRTEM measurements were prepared by dipping a carbon-coated copper grid into a suspension of ground samples in ethanol, which were pre-sonicated for 20 min. Field emission scanning electron microscope (FESEM) measurements were carried out with a JEOL JSM-6700F microscope. Samples were deposited on the surface of silicon wafer by dropping a suspension of ground samples in ethanol that was pre-sonicated for 20 min, and sputter coated for 2 cycles with gold. The accelerating voltage used was 5 kV with an electric current of 10 lA. Nitrogen adsorption–desorption isotherms were measured at 196 °C on a SA3100 surface area and pore size analyzer. Samples were degassed in a vacuum at 200 °C for 3 h prior to each measurement, while the lipase loaded samples were outgassed at 35 °C for 12 h. Brunauer-Emmett-Teller (BET) method was utilized to calculate the specific surface areas (SBET) from the adsorption data. Pore size distributions were derived from the adsorption branches of isotherms by using the Barrett-JoynerHalenda (BJH) model. Small-angle X-ray diffraction (SAXRD) patterns were recorded on a X’pert Pro powder X-ray diffractometer with Cu Ka radiation (40 kV, 40 mA), in reflection mode (2h = 0.5–5°) with a step size of 0.02° and a scan step time of 5 s. 2.4. Adsorption of lipase onto mesoporous supports Five milliliter of 50 mM sodium phosphate buffer (pH 6.0) were added to 40 mg mesoporous silica support in 20 mL capped vials. After the mixture was sonicated for 20 min, 5 mL of lipase stock solution with a concentration of 8 mg mL 1 in 50 mM phosphate buffer at pH 6.0 was added. The mixture was stirred by a magnetic stirrer at room temperature for different time periods (1–48 h). The supernatant was separated from the solid material by centrifugation (12000 rpm, 8 min, 4 °C). The amount of lipase immobilized onto the rod-like or vesicle-like silica was assayed by subtracting the amount in the supernatant liquid after adsorption from the amount of CRL present before addition of the mesoporous silicas, determined by UV–vis spectrophotometry at 258 nm; the supernatant liquid was isolated by centrifugation before the UV–vis mea-
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surements. The immobilized lipase was washed with phosphate buffer (pH 6.0, 10 mL) solution, dried overnight under vacuum at room temperature and then stored at 4 °C in a refrigerator. 2.5. Activity assays Activity of the immobilized lipase (with the lipase loading of 408 and 363 mg g 1 onto the rod-like and vesicle-like silica, respectively, after 16 h of adsorption) was determined by measuring the hydrolysis of tributyrin by the pH-stat method using a 310 Thermo Orion Perphect Meter [36]. In the hydrolysis reaction of tributyrin, butyric acid was produced as a by-product, which lowered pH of the hydrolysis mixture. The activity of the immobilized enzyme could then be determined by titrating the butyric acid with NaOH so as to maintain a constant pH. From the consumption of NaOH, the amount of butyric acid produced was obtained. The activity of the immobilized lipase was expressed as the percentage conversion of the tributyrin. Experiments with free lipase were performed by adding a desired amount of lipase. 2.6. Assay procedure A mixture of tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer saline (5 mL) was vigorously stirred at room temperature for 30 min, forming a tributyrin emulsion [38]. After pH of the emulsion was stabilized, the immobilized lipase (16 mg CRL) was added to the emulsion. The mixture was stirred for 6 h, and then centrifuged (12000 rpm, 8 min, 4 °C). The supernatant was titrated with 0.1 M sodium hydroxide solution so as to maintain a constant pH value. The volume of sodium hydroxide consumed was recorded and the activity of the immobilized CRL calculated. The activity of the free lipase (16 mg) was assayed by the same method. The same amounts (40 mg) of mesoporous silica were also used to measure the catalytic activity as blank experiments. Blank results showed that neither the mesoporous silicas nor the substrate consumed significant amounts of NaOH solution, indicating that assaying the activity of immobilized lipase by titration with NaOH is a viable procedure. 2.7. Thermal stability and operational stability Thermal stability experiments were first conducted by heating a solution of free lipase (16 mg CRL was solved in 2.5 mL phosphate buffer) or a suspension of immobilized lipase (immobilized lipase was dispersed in 2.5 mL phosphate buffer) in capped vials in a water bath at a desired temperature (23, 50, 60, 70 oC) for 30 min or at 60 oC for a desired amount of time (30, 60, 90 min). The solutions were allowed to cool down to room temperature. Afterwards, the mixture emulsion of tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (2.5 mL) was added for the hydrolysis of tributyrin. The operational stability of immobilized lipase was tested by repeating batch experiments (10 times) using the same substrate concentration, temperature and the reaction time as described above. 3. Results and discussion 3.1. Structures of the mesoporous rod-like and vesicle-like silicas Representative HRTEM images in Fig. 1 show clearly the rodlike and vesicle-like morphologies of the mesoporous silicas we obtained. Without TIPB, SBA-15 silica with a rod-like morphology of uniform size has been obtained similar to that prepared by Sayari et al. [3]. When a small amount of TIPB was added (TIPB to P123 molar ratio = 2.9:1), the morphology of the particles remained
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rod-like with hexagonal channels (Fig. 1a and b), but the pore diameter was increased from 9 nm to 11 nm. However when a large amount of TIPB was added, an unexpected drastic change occurred in the morphology from rod-like silica to vesicle-like or onion-like silica as shown in Fig. 1c and d. Almost all of the particles can be described as being multilamellar vesicles with a framework structure made of undulated silica layers ca. 5 nm thick with regularly spaced mesopores (12 nm); such an onion structure extends from the outer surface all the way inward to the center of the vesicle. As can be seen from the typical FESEM image presented in Fig. 2, many vesicle-like silica particles are ruptured obviously due to the calcination step, whereby gases inside the vesicles bustle out at the high temperatures. These breaches (20 nm) of vesicle-like silica can afford the entrance for the enzyme adsorption. 3.2. Lipase immobilization into mesoporous rod-like silica and vesiclelike silica The isoelectric point (pI or the pH at which the material has an equal number of positive and negative charges at its surface) of an enzyme is often considered for adsorption studies. The pI of CRL is 5.2 and hence, the protein is positively charged at a pH below pI and negatively charged at a pH above pI. On the other hand, the pI of the silica surface of SBA-15 support is about 3.7 ± 0.3 [22], Possible physical adsorption forces of CRL on mesoporous silica involved here include weak van der Waals interaction, hydrogen bonding interaction, and electrostatic interactions between the amino acid residues on the surface of CRL and silanol groups on the surface of the mesoporous materials [23], with the last being of the highest magnitude. When the solution pH is higher than the pI of CRL, the surface of the protein becomes negatively charged and therefore an electrostatic repulsion between the negatively charged CRL and the negatively charged silanol groups mesoporous silica support reduces the amount of CRL adsorption. At a pH near the isoelectric point, the net charge of the lipase is zero and hence the electrostatic interaction between the protein molecules is the lowest. The diminished electrostatic repulsion between the CRL molecules allows them to pack closer together onto the surface of the adsorbent in physical adsorption [23]. Proteins are known to show maximal adsorption when solution pH is near pI of protein [36], so we selected the solution with pH 6.0, which is near the pI of CRL. The lipase can enter the rod-like silica channels from their opened ends, while it can enter the vesicle-like silica curved channels from the breaches formed during calcination. The mesoporous rod-like and vesicle-like silicas were characterized by SAXRD and nitrogen adsorption before and after protein adsorption. The SAXRD patterns of the rod-like and the vesicle-like silicas before and after adsorption at an initial CRL concentration of 4 mg mL 1 at pH 6.0 are shown in Figs. 3 and 4, respectively. The rod-like silica before adsorption exhibits a strong reflection peak (1 0 0) and three small peaks (1 1 0, 2 0 0, 2 1 0) (Fig. 3a), characteristic of the hexagonal mesoporous structure. After lipase adsorption (Fig. 3b), the low angle reflection peak (1 0 0) can also be obtained, but the higher angle reflection peaks (1 1 0 and 2 0 0) are not observed, clearly reflecting the filling of the channels with the enzyme molecules. Similarly, the SAXRD patterns of the vesicle-like silica before (Fig. 4a) and after (Fig. 4b) adsorption of CRL show at least two well-resolved diffraction peaks (0 0 1 and 0 0 2) typical of the lamellar framework structure, but again, the higher-order reflection peak has suffered a significant intensity loss due to the enzyme adsorption. The fact that all of the original diffraction peaks are observed after enzyme adsorption indicates that both the rod-like and the vesicle-like silica maintain their longrange framework orders after the lipase loading [21,42]. Therefore we can conclude that the lipase molecules can be packed inside the
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Fig. 1. Representative HRTEM images of rod-like silica (a, b) and vesicle-like silica (c, d).
Fig. 3. SAXRD patterns of the rod-like silica before (a) and after (b) adsorption of CRL in pH 6.0 phosphate buffer solution for 16 h at an initial concentration of 4 mg mL 1 (CRL). Fig. 2. Representative FESEM image of vesicle-like silicas. The arrows point to the ruptured vesicle-like silicas.
mesopores without significantly affecting the structural integrity of the rod-like and vesicle-like silicas. The nitrogen adsorption–desorption isotherms of the rod-like and the vesicle-like silica and the resulting Barrett-Joyner-Halenda (BJH) pore size distributions determined from the corresponding adsorption branches before and after loading of CRL are shown in Figs. 5 and 6, respectively. One can see that the amount of nitrogen
adsorbed decreases markedly in the rod-like silica (from 687 cm3 g 1 to 472 cm3 g 1) and the vesicle-like silica (from 1028 cm3 g 1 to 703 cm3 g 1) upon CRL adsorption. Similar results have been reported for the adsorption of lysozyme and cytochrome c onto MCM-41 and SBA-15 [21,33]. The nitrogen adsorption– desorption isotherms of the rod-like silica before and after adsorption of CRL are of type IV with H1-type hysteresis loop that is characteristic of SBA-15. In contrast, the sorption isotherms of vesiclelike silica before and after adsorption of CRL are different from a typical type IV of SBA-15, e.g., they have a broad hysteresis loop
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Fig. 4. SAXRD patterns of vesicle-like silica before (a) and after (b) adsorption of CRL in pH 6.0 phosphate buffer solution for 16 h at an initial concentration of 4 mg mL 1 (CRL).
Fig. 6. N2 adsorption–desorption isotherms (a) and pore diameter distribution curves (b) of vesicle-like silica before (j) and after (d) lipase loading, and after lipase loading as well as after use for 10 runs of lipase catalysed hydrolysis of tributyrin (N).
Table 1 Structural parameters of the rod-like and vesicle-like silicas. TIPB:P123 molar ratio
SBET (m2 g
0 2.9/1 23.2/1
459 437 518
*
Fig. 5. N2 adsorption–desorption isotherms (a) and pore diameter distribution curves (b) of rod-like silica before (d) and after (j) lipase loading.
in the range of P/P0 = 0.51–0.99, which does not close until the saturation pressure is reached (Fig. 6a). Similar results were reported for the vesicle-like silica prepared by Lind et al. [13]. This result may be attributed to N2 filling the multilamellar mesopores of vesicular silica [10,13]. The vesicle-like silica before and after loading of lipase exhibit pore size distribution maxima centered at 12.3 and 11.1 nm, respectively (Fig. 6b). After 10 times of use for catalytic hydrolysis reactions, the vesicle-like silica immobilized lipase can maintain the shape of the isotherms of the original vesicle-like silica (Fig. 6a) and show pore size distribution maximum still centered at 11.1 nm (Fig. 6b). Moreover, the vesicle-like silica exhibits a much larger Brunauer-Emmett-Teller (BET) surface area SBET (520 m2 g 1) than that of the rod-like silica (440 m2 g 1) as well as a much larger total adsorption pore volume of 1.61 cm3 g 1 at a relative pressure of 0.99 (Table 1). Table 2 summarizes the textural properties of the rod-like and vesicle-like silicas after CRL loading and the data of catalytic reac-
Vp* (m3 g
1
)
1
)
0.95 1.09 1.61
WBJH (nm)
Morphology
d100spacing (nm)
9.2 11.1 12.3
Rod-like Rod-like Vesicle-like
9.7 10.6
d001spacing (nm)
11.8
The pore volume was obtained at relative pressure of 0.99.
tions for CRL immobilized in the vesicle-like silica. By comparing Table 2 and Table 1, one can see that the specific surface areas and the total pore volumes of the rod-like and vesicle-like silicas were drastically reduced after CRL adsorption. The specific surface area and the total pore volume of the rod-like silica decreased from 437 to 263 m2 g 1 and from 1.09 to 0.76 cm3 g 1, respectively, upon adsorption of CRL. More significantly, the adsorption of CRL led to the decrease of the specific surface area and the total pore volume of the vesicle-like silica from 518 to 235 m2 g 1 and from 1.61 to 1.11 cm3 g 1, respectively. The observed decrease in pore
Table 2 Structural parameters of the rod-like and vesicle-like silicas after CRL adsorption and after different numbers of runs for catalytic hydrolytic reactions. TIPB:P123 molar ratio
Number of reaction runs
SBET (m2 g
2.9:1 23.2:1 23.2:1 23.2:1
0 0 1 10
249 235 247 187
1
)
Vp (cm3 g 0.70 1.11 1.03 0.90
1
)
WBJH (nm) 11.1 12.3 11.1 11.1
228
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volume of 0.33 cm3 g 1 and specific surface area of 174 m2 g 1 for rod-like silica, and in pore volume of 0.50 cm3 g 1 and specific surface area of 283 m2 g 1 for vesicle-like silica after loading with CRL can be mainly attributed to the adsorption of CRL into the mesopores. However, other scenarios also need be considered. First, rod-like and vesicle-like silicas possess a large quantity of (ultra)micropores, therefore, some of the CRL molecules can block the pore mouths of the (ultra)micropores present in the silica walls. So, these micropores can no longer adsorb nitrogen, resulting in a reduction of the pore volume and specific surface area after CRL adsorption. Second, the increasing weight of rod-like and vesicle-like silicas after adsorption of CRL can also reduce the pore volume and specific surface area. Third, the tight packing of CRL molecules instead of adsorption in the mesopores may also decrease the pore volume and specific surface area. After 1 and 10 runs of catalytic hydrolytic reactions, the specific surface area and the total pore volume of the vesicle-like silica were reduced to 247 and 187 m2 g 1 and to 1.03 and 0.90 cm3 g 1, respectively. A possible explanation for this is a partial loss of the structural order of the vesicle-like silica after the 10 runs of the catalytic reactions. 3.3. Adsorption kinetics The adsorption kinetic curves of CRL into rod-like silica and vesicle-like silica in pH 6.0 solution at room temperature are shown in Fig. 7. We can find that the adsorption rate of CRL into rod-like silica is faster (reaching 193 mg g 1 within 1 h at room temperature) than that of vesicle-like silica (reaching 95 mg g 1 within 1 h at room temperature) and the amount of CRL immobilized into rodlike silica is higher (reaching 506 mg g 1 for 48 h at room temperature) than that of vesicle-like silica (reaching 476 mg g 1 for 48 h at room temperature). The significant difference in the adsorption rate and the adsorbed amount of CRL into rod-like silica and vesicle-like silica can be reasonably attributed to their different morphologies [31]. First, for rod-like silicas, there are at least two open entrances in the ends of each rod for lipase adsorption, whereas in the vesicle-like silicas, the entrances are limited to the breaches formed during calcination. Second, the mesochannel of rod-like silica is straight, whereas the mesochannel of vesiclelike silica is curved, which may influence the transport of the CRL. Therefore, the mesoporous morphologies appear to play an important role in their immobilization performance of enzyme. 3.4. Activity and stability of immobilized lipase
Our data show that the initial activity (the percentage conversion of tributyrin in the first run, 74%) of immobilized CRL on the vesicle-like silica is higher than that (66%) on the rod-like silica (Fig. 8). Moreover, after 10 successive batches of catalytic reactions, the hydrolysis activity of immobilized CRL on the vesicle-like silica reduced from 74% to 60%, whereas only 51% of the hydrolysis activity is retained for that on the rod-like silica. The decreased activities for the two immobilized CRL samples are mainly caused by lipase leakage owing to the multiple soaking and separation processes during the successive hydrolysis reactions. The better initial activity and operational stability of the vesicle-like silica immobilized CRL than the rod-like silica immobilized CRL is probably due to the unique flexual structure and the larger size of the mesochannels in the vesicle-like silica as well as the added inter-shell flexibility arising from the special vesicular structure. These factors would increase the affinity interaction between the silanol groups of vesicle-like silica and the lipase, resulting in a slower lipase leakage during the recycling process. The storage stability of immobilized CRL was also evaluated by running the catalytic reactions one time per week for 10 weeks (data not shown). The results are similar to those presented above, for immobilized CRL in both the rod-like and the vesicle-like silicas, demonstrating the good long-term storage stability of the immobilized lipase. The thermal stability of free and immobilized CRL was investigated and compared for catalytic hydrolysis of tributyrin by incubating the lipase for 30 min at different temperatures (Fig. 9) or
Fig. 8. Conversion of tributyrin versus the reaction run numbers. (j) Rod-like silica immobilized CRL; (d) Vesicle-like silica immobilized CRL. Hydrolysis conditions: tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (5 mL).
As mentioned above, one of the advantages of the immobilized enzymes is their reusability without compromising the activity.
Fig. 7. Adsorption kinetic curves of CRL into rod-like silica (j) and vesicle-like silica (d) in pH 6.0 solution at room temperature.
Fig. 9. Effect of thermal incubation temperature on conversion of tributyrin (incubation time: 30 min). (j) Rod-like silica immobilized CRL; (N) Free CRL; and (d) Vesicle-like silica immobilized CRL. Hydrolysis conditions: tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (5 mL).
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curvature of the mesochannels in the vesicle-like silica as well as the added inter-shell flexibility arising from the large size of the pores. Our study is the first to reveal the superior properties of the novel vesicle-like silica in protein adsorption and enzymatic catalysis and paves the way to the technological applications of this class of interesting materials. Acknowledgment We acknowledge supports from the Hong Kong University of Science and Technology (RPC06/07.SC03) and NNSFC-RGC (N_HKUST604/04) and RGC (604206) administrated by the UGC of Hong Kong. Fig. 10. Effect of incubation time on conversion of tributyrin (incubation temperature 60 °C). (j) Rod-like silica immobilized CRL; (N) Free CRL; and (d) Vesicle-like silica immobilized CRL. Hydrolysis conditions: tributyrin (0.4 g), water (9.8 mL), and pH 7.0 phosphate buffer (5 mL).
incubating the lipase at 60 °C for different times (Fig. 10). The catalytic activity of the vesicle-like silica immobilized CRL for hydrolysis of tributyrin is 67% and 46% when incubated at temperature of 60 °C for 30 min and 90 min, respectively, whereas it is merely 46% and 32% with the rod-like silica immobilized CRL at the same condition. It can also be seen that the vesicle-like silica immobilized CRL shows a higher thermal stability than the free CRL. In contrast, the rod-like silica immobilized CRL exhibits a reduced hydrolysis activity after the thermal treatment relative to that of the free lipase. Similarly, Hudson et al. [22] reported that cytochrome c immobilized onto SBA-15 showed a higher intrinsic activity but lower thermostability than free cytochrome c. We can conclude that the CRL immobilized onto the vesicle-like silica shows resistance to leaching and an enhanced activity compared to the rod-like silica immobilized CRL. A likely scenario is that the active sites of the immobilized lipase into the rod-like silica are more susceptible to heat denaturation. The higher stability and activity of the lipase immobilized in vesicle-like silicas makes them an excellent candidate for a new bioimmobilization host. 4. Conclusions The vesicle-like silica we have developed possesses large uniform interlamellar pores and a completely multilamellar structure in the whole vesicular spheres. These features are advantageous for applications in many technological areas involving adsorption of large molecules such as proteins and enzyme catalysis. For comparative studies, lipase from Candida rugosa (CRL) has been immobilized in the rod-like and vesicle-like silicas by physical adsorption. The SAXRD and N2 adsorption–desorption data after CRL adsorption reveal that the mesostructure can be maintained and the CRL molecules are efficiently adsorbed inside the mesopores of the rod-like silica and the vesicle-like silicas. The catalytic activity, thermostability, and operational stability of the immobilized lipase were measured through the hydrolysis of tributyrin in phosphate buffer solution. After 10 runs of catalytic hydrolysis of tributyrin, the catalytic hydrolysis activity of immobilized CRL on the vesicle-like silica is reduced from 74% to 60%, whereas more reduction from 66% to 51% is found for the immobilized CRL on the rod-like silica. When incubated at temperature of 60 °C for 30 min and 90 min, the catalytic activity of the vesicle-like silica immobilized CRL for hydrolysis of tributyrin is, respectively, 67% and 46%, whereas it is merely 46% and 32% with the rod-like silica immobilized CRL. The lipase immobilized into the vesicle-like silica shows substantially higher activity, reusability and thermal stability compared to that into the rod-like silica due probably to the unique
References [1] D. Zhao, Q. Huo, J. Feng, B.F. Chmelka, G.D. Stucky, J. Am. Chem. Soc. 120 (1998) 6024. [2] D. Zhao, J. Feng, Q. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka, G.D. Stucky, Science 279 (1998) 548. [3] A. Sayari, B.-H. Han, Y. Yang, J. Am. Chem. Soc. 126 (2004) 14348. [4] J.D. Rimer, J.M. Fedeyko, D.G. Vlachos, R.F. Lobo, Chem. Eur. J. 12 (2006) 2926. [5] S. Che, K. Lund, T. Tatsumi, S. Iijima, S.H. Joo, R. Ryoo, O. Terasaki, Angew. Chem. Int. Ed. 42 (2003) 2182. [6] N.E. Botterhuis, Q. Sun, P.C.M.M. Magusin, R.A.V. Santen, N.A.J.M. Sommerdijk, Chem. Eur. J. 12 (2006) 1448. [7] F. Hoffmann, M. Cornelius, J. Morell, M. Froba, Angew. Chem. Int. Ed. 45 (2006) 3216. [8] P.T. Tanev, T.J. Pinnavaia, Science 271 (1996) 1267. [9] P.T. Tanev, Y. Liang, T.J. Pinnavaia, J. Am. Chem. Soc. 119 (1997) 8616. [10] S.S. Kim, W. Zhang, T.J. Pinnavaia, Science 282 (1998) 1302. [11] S.S. Kim, Y. Liu, T.J. Pinnavaia, Micropor. Mesopor. Mater. 44–45 (2001) 489. [12] A.J. Karkamkar, S.S. Kim, S.D. Mahanti, T.J. Pinnavaia, Adv. Funct. Mater. 14 (2004) 507. [13] A. Lind, B. Spliethoff, M. Linden, Chem. Mater. 15 (2003) 813. [14] H.-P. Hentze, S.R. Raghavan, C.A. McKelvey, E.W. Kaler, Langmuir 19 (2003) 1069. [15] R.K. Rana, Y. Mastai, A. Gedanken, Adv. Mater. 14 (2002) 1414. [16] J.G.C. Shen, J. Phys. Chem. B 108 (2004) 44. [17] J. Sun, D. Ma, H. Zhang, C. Wang, X. Bao, D.S. Su, A. Klein-Hoffmann, G. Weinberg, S. Mann, J. Mater. Chem. 16 (2006) 1507. [18] Y.Q. Yeh, B.C. Chen, H.P. Lin, C.Y. Tang, Langmuir 22 (2006) 6. [19] J. Liu, Q. Yang, L. Zhang, D. Jiang, X. Shi, J. Yang, H. Zhong, C. Li, Adv. Funct. Mater. 17 (2007) 569. [20] J.F. Diaz, K.J. Balkus, J. Mol. Catal. B 2 (1996) 115. [21] A. Vinu, V. Murugesan, O. Tangermann, M. Hartmann, Chem. Mater. 16 (2004) 3056. [22] S. Hudson, E. Magner, J. Cooney, B.K. Hodnett, J. Phys. Chem. B 109 (2005) 19496. [23] H.H.P. Yiu, P.A. Wright, J. Mater. Chem. 15 (2005) 3690. [24] D. Moelans, P. Cool, J. Baeyens, E.F. Vansant, Catal. Commun. 6 (2005) 307. [25] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Micropor. Mesopor. Mater. 44–45 (2001) 755. [26] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Chem. Mater. 12 (2000) 3301. [27] Y. Han, S.S. Lee, J.Y. Ying, Chem. Mater. 18 (2006) 643. [28] Y. Wang, F. Caruso, Chem. Mater. 17 (2005) 953. [29] J. Deere, E. Magner, J.G. Wall, B.K. Hodnett, J. Phys. Chem. B 106 (2002) 7340. [30] J. Aburto, M. Ayala, I. Bustos-Jaimes, C. Montiel, E. Terres, J.M. Dominguez, E. Torres, Micropor. Mesopor. Mater. 83 (2005) 193. [31] J. Lei, J. Fan, C. Yu, L. Zhang, S. Jiang, B. Tu, D. Zhao, Micropor. Mesopor. Mater. 73 (2004) 121. [32] A. Katiyar, L. Ji, P. Smirniotis, N.G. Pinto, J. Chromatogr. A 1069 (2005) 119. [33] A. Vinu, V. Murugesan, M. Hartmann, J. Phys. Chem. B 108 (2004) 7323. [34] J.L. Blin, C. Cerardin, C. Carteret, L. Rodehuser, C. Selve, M.J. Stebe, Chem. Mater. 17 (2005) 1479. [35] K. Han, Z. Wu, J. Lee, I.S. Ahn, J.W. Park, B.R. Min, K. Lee, Biochem. Eng. J. 22 (2005) 161. [36] A. Salis, D. Meloni, S. Ligas, M.F. Casula, M. Monduzzi, V. Solinas, E. Dumitriu, Langmuir 21 (2005) 5511. [37] E. Dumitriu, F. Secundo, J. Patarin, I. Fechete, J. Mol. Catal. B: Enzymatic 22 (2003) 119. [38] H. Ma, J. He, D.G. Evans, X. Duan, J. Mol. Catal. B: Enzymatic 30 (2004) 209. [39] Z.Z. Chen, Y.M. Li, X. Peng, F.R. Huang, Y.F. Zhao, J. Mol. Catal. B: Enzymatic 18 (2002) 243. [40] M. Mureseanu, A. Galarneau, G. Renard, F. Fajula, Langmuir 21 (2005) 4648. [41] G. Zhou, Y. Chen, J. Yang, S. Yang, J. Mater. Chem. 17 (2007) 2839. [42] W. Hammond, E. Prouzet, S.D. Mahanti, T.J. Pinnavaia, Micropor. Mesopor. Mater. 27 (1999) 19.