Article pubs.acs.org/JPCC
Hybrid Periodic Mesoporous Organosilicas (PMO-SBA-16): A Support for Immobilization of D-Amino Acid Oxidase and Glutaryl-7-amino Cephalosporanic Acid Acylase Enzymes Bendaoud Nohair,† Phan thi Hong Thao,‡ Vu Thi Hanh Nguyen,‡ Phi Quyet Tien,*,‡ Dang Tuyet Phuong,§ Le Gia Hy,‡ and Serge Kaliaguine*,† †
Department of Chemical Engineering, Laval University, Quebec (Quebec), Canada G1V 0A6 Institute of Biotechnology, Vietnam Academy of Science and Technology § Institute of Chemistry, Vietnam Academy of Science and Technology ‡
S Supporting Information *
ABSTRACT: This study examined the adsorption and stability of D-amino acid oxidase (DAAO) and glutaryl-7-amino cephalosporanic acid acylase (GL-7-ACA acylase) enzymes using two different types of periodic mesoporous organosilicas PMO-SBA-16 synthesized from 1,2bis(trimethoxysilyl)ethane (BTME) and 1,4-bis(triethoxysilyl)benzene (BTEB). Very high loading, specific enzymatic activities, and stabilities have been reached by proper optimization of mesopore structure and morphology.
1. INTRODUCTION The utilization of enzymes as biocatalysts has become an important avenue in chemical and pharmaceutical industries to prepare biochemical products, biosensors, and drugs.1−5 Enzymatic reactions are green and environmentally sustainable processes in the production of a wide range of natural products. The catalytic efficiency of enzymes is high under normal mild reaction conditions. However, the major drawbacks of enzymes for industrial applications are their low long-term stability, the difficult recovery, and product contamination. One of the approaches to resolve these difficulties is to immobilize enzymes on solid surfaces such as mesoporous silica to provide stable and active biocatalysts under process conditions. The immobilization of biomolecules onto mesoporous silica materials is quite useful for practical applications due to the potential to improve their stability and to retain their activity under extreme conditions (temperature or pH). Also, the enzyme molecules are fully dispersed without aggregation or the possibility of interacting with any external interface due to their immobilization inside the porous structure. Recent reports show that ordered mesoporous materials are efficient as supports for the immobilization of enzymes.6−9 When a mesoporous material is used as support, the large pores facilitate transport of substrate and product.10 Therefore, the pore size, morphology, particle size, surface area, and surface chemistry are five major factors that affect immobilization on mesoporous silicas.11,12 Deere et al.13−15 showed that the amount and stability of adsorbed protein depended on the mesoporous silica pore diameter, isoelectric point, degree of hydrophilicity/hydrophobicity, and type of surfactant template used to synthesize the mesoporous silicas. The most common methods of enzyme immobilization include physical or chemical adsorption16and physical entrapment.17 The principal driving forces in the adsorption of © 2012 American Chemical Society
biomolecules onto porous hosts are electrostatic, hydrogen bonding, and weak van der Waals interactions.6 The “nucleus” of 7-amino cephalosporanic acid (7-ACA) is an important precursor for the production of semisynthetic antibiotics18 of the cephem group, which are widely used for effective treatment of infectious diseases. There are two different pathways of 7-ACA production from natural cephalosporin C (CPC): (i) chemical conversion (processed at −70 °C) utilizing large quantities of toxic and polluting reagents19 such as trimethylchlorosylane, dimethylaniline, and PCl5; (ii) enzymatic synthesis, in which two enzymes, D-amino acid oxidase (DAAO) and glutaryl 7-aminocephalosporanic acid acylase (GL-7-ACA acylase), are used. The latter pathway has proved to be highly effective and environmentally friendly; therefore, it is a promising solution for industry (Scheme 1). However, improvement of DAAO and GL-7-ACA acylase activities and efficient application of both catalysts in the conversion of CPC to 7-ACA still remain significant challenges. We report herein the use of periodic mesoporous organosilicas, namely, PMOs20−22 as supports. Their synthesis was realized by the condensation of bridged organosilica precursors of the type (R′O)3Si−R−Si(R′O)3 with R′ being methoxy or ethoxy groups and R being an organic bridge. These mesoporous organosilicas represent a promising class of organic−inorganic nanocomposite materials with a large pore size (2−14 nm), in which the organic and inorganic moieties are covalently linked and homogeneously distributed at a molecular level. Compared with their mesoporous silica counterparts, the unique hydrophilic−hydrophobic frameworks of PMOs can be easily adjusted through incorporation of Received: December 5, 2011 Revised: April 6, 2012 Published: April 6, 2012 10904
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Scheme 1. Enzymatic Conversion of CPC to 7-ACA by Using Two Enzymes, DAAO and GL-7-ACA Acylase
hydrothermal treatment under static conditions at temperature varying from 80 to 130 °C for 1 or 2 days. The product was filtered and air-dried, followed by surfactant removal by calcination at 550 °C during 6 h for SBA-16 and Soxhlet extraction with ethanol for 48 or 96 h for PMO-SBA-16. The final material was dried at 60 °C overnight. The prepared materials are referred to as PMO-SBA16-T(t) where PMO represents the precursor and T and t represent the temperature and time of hydrothermal treatment, respectively. 2.2. Immobilization Procedure of Enzymes on Supports. 2.2.1. Preparation of DAAO. DAAO used in the present research was biosynthesized by the recombinant bacterium Escherichia coli BL21(DE3) [pET 22b-daao], which was generated in a previously reported work.54 To produce DAAO, 5.0 mL of recombinant E. coli preculture was inoculated into 50 mL of Luria−Bertani (LB) medium (tryptone, 10 g·L−1; yeast extract, 5 g·L−1; NaCl, 10 g·L−1) supplemented with ampicillin 100 μg/mL. The culture was then incubated on a rotary shaker at 37 °C until optical density (OD) at 600 nm reached 0.6 unit and then induced with 0.4 μmol of isopropylβ-D-thio-galactoside (IPTG) for 3 h before harvest. The average activity of free DAAO used for further experiments in enzyme immobilization was 11.5 U/mg of protein. Immobilization of DAAO on Mesomaterials. Immobilization of DAAO was performed according to Salis et al.31 as follows: 100 mg of mesoporous material was suspended in 4 mL of DAAO solution at pH 8.0 at 25 °C under stirring for 7 h at 200 rpm. The suspension was then centrifuged, and the enzyme immobilized on functionalized material was washed with phosphate buffer pH 8. The remaining protein in supernatant was measured by a previously described method.54 The immobilized DAAO was stored in phosphate buffer pH 8.0 at 4 °C. Activity Assays of Free and Immobilized DAAO. The DAAO activity was determined by using D-alanine as the substrate, in which enzymatic reaction was performed at 30 °C in 50 mM phosphate buffer (pH 8.0) as described previously.32 One unit of DAAO activity was defined as the amount of enzyme that oxidized 1.0 μmol of D-alanine per minute under the above-mentioned conditions. 2.2.2. GL-7-ACA Acylase Enzyme. Preparation of GL-7-ACA Acylase. GL-7-ACA acylase used in the present research was
different kinds of organic groups in the mesoporous framework without pore blocking.23 In addition, incorporation of organic bridging groups improves the stability of the resulting materials.24 This study examined the effect of using PMOs-SBA-16 as novel supports with different pore sizes (from 8 to 10 nm) and with different frameworks on the DAAO and GL-7-ACA acylase enzyme adsorption. The PMOs-SBA-16 materials were synthesized by using 1,2-bis(trimethoxysilyl)ethane (BTME) and 1,4-bis(triethoxysilyl)benzene (BTEB) precursors in the presence of triblock copolymer F127 as a template, under varying conditions affecting the pore size distribution. For the sake of comparison, a pure-silica SBA-16 material was prepared using tetraethyl orthosilicate (TEOS) as a precursor inorganic framework material. The activity of immobilized enzymes was tested in the conversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid (GL-7-ACA) (Scheme 1).
2. EXPERIMENTAL SECTION 2.1. Preparation of Supports: SBA-16 and PMOs-SBA16. SBA-16 Material. The synthesis of pure-silica SBA-16 was similar to that reported recently by Ryoo et al.25,26 and Kaliaguine et al.27,28 An amount of 1.07 g of block copolymer F127 was dissolved with stirring in a solution of 51.44 g of distilled water and 2.12 g of concentrated hydrochloric acid (HCl, Fischer 36.5−38.0%) at room temperature. The solution was heated to 45 °C before adding 3.21 g of the cosurfactant butanol. After about 1 h, 5.09 g of TEOS was added to the solution. The molar composition of the reaction mixture TEOS/F127/BuOH/HCl/H2O is 1.0/ 0.003/1.78/0.88/119. PMOs-SBA-16 Material. The synthesis of the PMO-SBA-16 sample was similar to that reported by Guo et al.29,30 Amounts of 1.8 g of F127 and 19.9 g of NaCl were dissolved in 30 g of distilled water and 90 g of 2.0 M HCl (Fischer 36.5− 38.0%) solution with stirring at room temperature. Then, 4.8 g of BTME (ethane bridging groups) or 7.15 g of BTEB (phenylene-bridging groups) were added to the homogeneous solution, while the stirring continued for 24 h. In all cases, the resulting mixture was stirred at the desired temperature (45 °C for SBA-16 and 40 °C for PMOs-SBA-16) for 24 h and then transferred to a Teflon bottle to allow for 10905
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Figure 1. X-ray diffraction patterns for (A) BTME-SBA16 supports (a, BTME-SBA16-130(2d); b, BTME-SBA16-130(1d); c, BTME-SBA16100(2d); d,e, BTME-SBA16-100(1d), respectively, extracted for 96 and 48 h; f, BTME-SBA16-80(1d)) (successive samples offset for clarity) and (B) BTEB-SBA16 supports (a, BTEB-SBA16-100(1d); b, BTEB-SBA16-80(1d)).
obtained at P/P0 between 0.1 and 0.2. The total pore volume was estimated from the volume of N2 adsorbed at a relative pressure of P/P0 = 0.99. Nonlocal density functional theory (NLDFT) analyses were also performed to evaluate surface area, pore volume, and pore size. For these analyses, the kernel of NLDFT equilibrium capillary condensation isotherms of N2 at −196 °C on silica was selected for the model isotherms (using the desorption model and assuming cylinder/sphere pores). Powder XRD spectra were recorded using a Bruker D4 X-ray diffractometer with nickel-filtered Cu Kα radiation (λ = 1.5418 Å). The tube voltage was 40 kV, while the current was 40 mA. Diffraction patterns were recorded with scan step of 0.02° for 2 theta between 0.5° and 5°. Transmission electron micrographs (TEM) were obtained using a JEOL JEM 1230 microscope operated at 80 kV. Scanning electron microscopy (SEM) images were recorded using a JEOL 840A microscope operated at an accelerating voltage of 10−20 kV. All SEM images reported here are representative of the corresponding materials.13C MAS NMR spectra were recorded at room temperature using a Bruker ASX 300 spectrometer. Adamantane was used as the external reference for 13C MAS NMR analysis.
synthesized by bacterial strain Citrobacter f reundii Hg32 as previously described.55 The preculture of strain Hg32 was cultivated in optimized medium (casein hydrolysate, 20 g·L−1; yeast extract, 5.0 g·L−1; natri glutamate, 5 g·L−1; corn steep liquor, g·L−1; glutaric acid, 1.0 g·L−1; pH 8,0) on a rotary shaker at 220 rotations per min (rpm) and 30 °C for 30 h. The cells were harvested by centrifugation at 10 000 rpm for 4 min and then disrupted by sonication (20 kHz for 30 s). Enzyme GL-7ACA acylase in supernatant of cell lysate was obtained by centrifugation at 12 000 rmp for 10 min. The average activity of free GL-7-ACA acylase used for further experiments in enzyme immobilization was 3.02 U/mg of protein. Assay of Free GL-7-ACA Acylase Activity. Activity of free GL-7-ACA acylase activity was measured by the method described by Shibuya et al.53 Immobilization of GL-7-ACA Acylase. The procedure for immobilization of GL-7-ACA acylase on mesostructured materials was performed as follows: 100 mg of mesostructured materials was added to 3 mL of GL-7-ACA acylase solution (diluted to 5.8 U/mL). After stirring the mixture at 150 rpm for 3 h, the immobilized enzyme was collected by centrifugation at 2000 rpm for 5 min and then washed twice with 100 mM phosphate buffer. Activity Assay of GL-7-ACA Acylase. The immobilized enzyme was incubated at 37 °C for 30 min in the presence of 1% (w/v) substrate GL-7-ACA. The reaction was stopped using an aqueous solution of 20% (v/v) acetic acid and 0.05 N NaOH. Then, p-dimethylaminobenzaldehyde (PDAB, 0.5% w/ v in methanol) was added to the mixture. The products in the supernatant were determined by UV−vis spectrophotometry at 415 nm. One unit of GL-7-ACA acylase activity was defined as the amount of enzyme that produced 1 μmol of 7-ACA per min at 37 °C, pH 8. 2.3. Supports Characterization. Nitrogen adsorption measurements were performed at −196 °C using a Quantachrome Autosorb1 volumetric adsorption analyzer. Before the measurements, the samples were outgassed at 120 °C for 5 h under vacuum, until a residual pressure of 10−6 mbar was reached. The Brunauer−Emmett−Teller (BET) equation was used to calculate the surface area SBET from adsorption data
3. RESULTS AND DISCUSSION The XRD patterns of solvent-extracted BTME-SBA16 (Figure 1A) and BTEB-SBA16 (Figure 1B) support materials after hydrothermal treatment are shown in Figure 1. In both cases, XRD patterns show a very strong peak at 2θ varying between 0.64° and 0.74° and a minor peak at 2θ ≈ 1.04°. These peaks can be indexed as (110), (200) reflections of 3D cubic Im3m symmetry, similar to that reported for the silica-based mesoporous SBA-1633,34 and PMOs-SBA-16.29,30 However, for all PMOs-SBA16, the increase of the hydrothermal treatment time and temperature resulted in structural disordering; the (110) diffraction peak shifted to a larger 2θ angle especially with BTEB (Figure 1B) accompanied by changes in the relative intensities (decrease); and also (200) reflection was difficult to resolve while the higher reflections tend to increase. This observation suggests that a high ratio of wall thickness 10906
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TEM and SEM. The effects of temperature and time of hydrothermal treatment on morphologies of periodic mesoporous organosilica materials PMO-SBA-16 were investigated by TEM and SEM measurements as shown in Figures 2 and 3, respectively. Both the BTME-SBA16 and BTEB-SBA16 support materials clearly show well-ordered domains of three-dimensional cubic mesostructure in their TEM images (Figure 1), similar to the ones reported for the silica-based mesoporous TEOS-SBA-16. The TEM estimated pore diameters are in good agreement with those obtained from the nitrogen adsorption measurements. The morphologies of the template-free samples after hydrothermal treatment are presented in Figure 3. It is apparent that both BTME-SBA16 aged at 100 and 130 °C during 2 and 1 day, respectively, were shown to have almost spherical particle morphologies with diameters varying from 0.5 to 5 μm with spheres for the sample treated at 130 °C being generally larger. This suggests that the aging time and temperature affect the macrostructure of the mesoporous materials. In the case of BTEB-SBA16 materials, the shapes are not very well-defined. Solid-State NMR. Solid-state 13C CP MAS NMR spectrum of the extracted BTME-SBA16-100(1D) is shown in Figure 4A. The PMO material displayed a strong resonance at 4.9 ppm, which can be assigned to the bridging ethane moiety (Si− CH2CH2−Si).38,39 In the case of extracted BTEB-SBA16100(1D) (Figure. 4B), the carbon atoms of phenylene groups were found at 133.6 ppm, indicating that the phenylene groups40,41 (Si−(C6H4)−Si) in the material are intact. The additional two weak signals at 13.9 and 53.3 ppm were due to the ethoxy groups Si−OCH2CH3 formed during the surfactant removal process by ethanol. Nitrogen Adsorption−Desorption. All samples of BTME-SBA16 and BTEB-SBA16 show a type IV isotherm (Figures 5A and 6A) with a sharp capillary condensation step at high relative pressures, typical of the highly organized mesostructured materials with significant differences in pore diameters (Figure. 6B). The structural parameters derived from the isotherms are reported in Table 1. It can be seen from Table 1 that, in the case of BTME-SBA16, an increase in aging temperature from 80 to 130 °C led to a slight increase in BET specific surface area and total pore volume. However, the pore diameter of the materials is not influenced by the time and temperature of aging (good agreement with XRD analysis, Figure 1A). In the case of BTEBSBA-16, as the aging temperature increases from 80 to 100 °C, the pore diameter and the surface area of the material shifts from 8.1 to 10.4 nm and from 180 to 405 m2 g−1, respectively (Table 1). The observed trends are in a fairly good agreement with those observed on SBA-16 pure silicas.27 At present, particular interest is focused on large-pore PMO materials with different frameworks for the immobilization and encapsulation of large molecules.42 Thus, this work on PMOSBA-16 synthesized with various structural parameters, hydrophobic character (organic bridges), and external morphologies (size of the spheres) is of significant interest in the biocatalysis field. DAAO Enzyme Adsorption and Activity. The loading and activities of DAAO enzyme immobilized on mesoporous PMO-SBA-16 and TEOS-SBA-16 with different pore sizes and morphologies are shown in Table 2. In our case, the DAAO enzyme (11.2 × 4.5 × 3.1 nm)43 fits well within the size of the
Figure 2. TEM image of (A) BTME-SBA16-80(1D), (B,C) BTMESBA16-100(1D), respectively, extracted for 48 and 96 h, and (D) BTEB-SBA16-80(1D).
Figure 3. SEM image of (A) BTME-SBA16-100(2D), (B) BTMESBA16-130(1D), (C) BTEB-SBA16-80(1D), and (D) BTEB-SBA16100(1D).
Figure 4. 13C NMR spectra of (A) BTME-SBA16-100(1D) and (B) BTEB-SBA16-100(1D). (* Signals due to spinning side bands.)
versus pore size produces lower relative intensities for higherorder diffractions, as suggested previously.27,35−37 10907
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Figure 5. (A) N2 adsorption−desorption and (B) NLDFT pore size distributions calculated from the adsorption branches of the isotherms of BTME-SBA16 supports (a, BTME-SBA16-130(2d); b, BTME-SBA16-130(1d); c, BTME-SBA16-100(2d); d,e, BTME-SBA16-100(1d), respectively, extracted for 96 and 48 h; f, BTME-SBA16-80(1d)). (Successive samples offset for clarity.)
Figure 6. (A) N2 adsorption−desorption and (B) NLDFT pore size distributions calculated from the adsorption branches of the isotherms of BTEB-SBA16 supports. (Successive samples offset for clarity.)
Table 1. Physical Properties of Mesoporous Silicas TEOS-SBA-16 and Organosilicas PMOs-SBA-16a materials
removal surfactant
SBET, m2 g−1
SDFT, m2 g−1
VtDFT, cm3 g−1
VmDFT, cm3 g−1
WBJH, nm
WDFT, nm
TEOS-SBA16-100(1D) TEOS-SBA16-100(2D) BTME-SBA16-80(1D) BTME-SBA16-100(1D)-ex48 BTME-SBA16-100 (1D)-ex96 BTME-SBA16-100(2D) BTME-SBA16-130(1D) BTME-SBA16-130(2D) BTEB-SBA16-80(1D) BTEB-SBA16-100(1D)
Cal-550 °C Cal-550 °C Ex-96 h Ex-48 h Ex-96 h Ex-48 h Ex-48 h Ex-48 h Ex-96 h Ex-48 h
735 1062 513 515 681 453 627 622 180 405
636 874 489 565 601 376 545 561 131 323
0.57 0.80 0.35 0.35 0.45 0.33 0.43 0.40 0.15 0.37
0.079 0.095 0.10 0.11 0.10 0.05 0.08 0.10 0.00 0.01
6.9 7.3 6.9 6.7 6.8 6.9 6.7 6.9 4.8 6.8
9.7 10.9 10.1 9.8 10.1 10.1 9.8 10.1 8.1 10.4
a SDFT is the specific surface area; VtDFT is the total pore volume; VmDFT is the micropores volume; WDFT is the mesopores diameter, calculated by the DFT method using the kernel of NLDFT equilibrium capillary condensation isotherms of N2 at 77 K on silica, adsorption branch. Cal: calcination; Ex: extraction.
pores (Table 1) that are at least 8.1 nm in diameter. So, for all supports the enzyme can be incorporated inside the spherical cages as well as on the external surface of the particles. This good agreement between host and guest dimensions has been suggested to improve the enzymatic activity and stability.44,45 The results reported in Table 2 show that the enzymatic activity and specific activity of all PMO-SBA-16 organosilicas are significantly higher than those of TEOS-SBA-16 pure-silica
materials. The specific activity of enzyme and protein loaded on BTME-SBA16-100(1D)-ex48 is however the highest. The specific DAAO activity reached with this support (4.48 U/ mg) may be compared with the one of the free DAAO enzyme (11.5 U/mg). Thus, in spite of the mass transfer limitation associated with the supported enzyme, close to 40% of the enzyme-specific activity is retained. The DAAO immobilized on BTME-SBA16-100(1D)-ex48 support displays an enzymatic 10908
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difference between the periodic mesoporous organosilicas (PMO) and pure silicas SBA-16 is the presence of a partially organic framework with a difference in the surface chemistry of the adsorbent and its hydrophilicity/hydrophobicity,9 which seems to affect drastically the enzyme activity. The observed higher loading and activity of DAAO on BTME-SBA16 compared with BTEB-SBA16 might be explained by the low surface area and pore volume caused by the larger and more flexible organic bridging group BTEB in the framework, resulting in some reduction of enzyme diffusion and access of the substrates to the active site of the immobilized enzymes inside the mesopore. With BTEB-SBA16 supports, the amount of DAAO loading is 1.7 times higher at 100(1D) than that at 80(1D). This is related to the smaller pore size of the latter material (BTEB-SBA16-80(1D)). There is therefore indeed some relationship between the pore size of the materials and the enzyme loading process as previously reported.13,46,47 Stability of DAAO on Supports. In biocatalysis, increased operational stability of immobilized enzymes is important for achieving cost benefits. The stability of DAAO immobilized on the different support materials stored at 4 °C in phosphate buffer solution (pH 8) is listed in Table 3A. It can be seen that the enzymatic activity of DAAO immobilized on PMO-SBA16 supports is only slightly decreasing with time with 74−87.5% of the original activity being retained after 20 days for five of the supports tested. The retention of activity was lower on TEOSSBA16 which confirms a high affinity of DAAO to PMO supports, and some DAAO enzyme may thus be trapped inside the cages of the mesoporous structure. The significant loss of enzymatic activity of the DAAO immobilized on the TEOSSBA16 support is likely due to enzyme leaching from the support during the reaction. The stability in several of the PMO supports is even found higher than that of the free DAAO enzyme (see Table 3B). The interactions of enzyme with the surface of PMO-SBA-16 materials can lead to enhancement of the operational stability of the immobilized enzyme. Enhanced stability is also attributed to the stabilizing effect of the support matrix, which prevents the conformational changes of enzymes. BTME and BTEB groups decrease the size of pore aperture, causing more entrapping of the DAAO enzymes inside the mesopores8,48 which reduces the amount of leaching and allows the immobilized enzyme to be reused. The low leaching reflects the strong interaction between the enzymes DAAO and the organosilica supports. GL-7-ACA Acylase Enzyme Adsorption, Activity, and Stability. The loading of enzyme and specific and enzymatic activity of GL-7-ACA acylase immobilized on different support materials are listed in Table 4. Among the four PMO-SBA-16, three have loadings higher than 50 mg/g which is higher than the values obtained with silica SBA-16 samples. The only exception is sample BTME-SBA-16-100(2D) which shows the smallest loading of all samples. The specific activity of the same sample is however by far the highest: the 2.03 U/mg observed value represents 67% of the specific activity of the free GL-7ACA enzyme (3.02 U/mg). Since this difference is associated with mass transfer limitation within the pore lattice of the support this result suggests that the lower specific activity observed with all other samples is related to increased mass transfer resistance due to the presence of higher enzyme loading in the pore lattice. The overall activity of the BTME-SBA-16-100(2D) sample is also relatively high (25.9 U/g) being only surpassed by the
Table 2. Immobilized DAAO on Mesoporous Silicas TEOSSBA-16 and Organosilicas PMOs-SBA-16a supports
protein loading (mg/g)
enzyme protein loading yield (%)
DAAO specific activity (U/ mg)b
DAAO activity (U/g)c
46.1
24.3
0.89
40.9
53.8
28.4
0.71
38.3
25.9
13.7
4.09
105.9
68.6
36.2
4.48
307.3
34.4
18.1
2.32
79.8
53.8
28.4
2.11
113.7
39.5
20.8
2.41
95.1
50.9
26.8
3.74
190.3
52.1
27.5
1.28
66.8
26.9
14.2
2.18
58.7
46.5
24.5
2.41
112.1
TEOS-SBA16100(2D) 1test TEOS-SBA16100(2D) 2test BTME-SBA1680(1D) BTME-SBA16100(1D)ex48 BTME-SBA16100(1D)ex96 BTME-SBA16100(2D) 1test BTME-SBA16100(2D) 2test BTME-SBA16130(1D) BTME-SBA16130(2D) BTEB-SBA1680(1D) BTEB-SBA16100(1D) a
Note: Free DAAO initial activity: 54.66 U/mL and initial protein 4.738 mg/mL. bEnzyme activity per 1 mg of protein. cEnzyme activity per 1 g of (dry weight) catalyst.
Table 3. (A) Stability of DAAO on Mesoporous Silicas TEOS-SBA-16 and Organosilicas PMOs-SBA-16 and (B) Relative Activity Time Variation of Free DAAO % (A)
variation of enzymatic activity with time (U/g at dried weight) 8 days
13 days
14 days
20 days
29.6 40.5
-
28.2 19.7
-
-
38.4
28.8
3.65
-
0
0
106.0
89.3
50.3
-
33.1
-
307.4
288.8
-
272.3
-
261.5
79.8
79.4
28.3
-
18.6
-
113.8
107.8
-
85.9
-
83.8
95.1
82.7
55.4
-
33.8
-
190.3
170.4
165.5
160.1
66.8
55.0
53.3
52.1
58.7
52.1
112.1
111.4
supports
1 day
3 days
TEOS-SBA16 (VN) TEOS-SBA16 -100(2D) 1es TEOS-SBA16100(2D) 2es BTME-SBA1680(1D) BTME-SBA16100(1D)-ex48 BTME-SBA16100(1D)-ex96 BTME-SBA16 -100(2D) 1es BTME-SBA16100(2D) 2es BTME-SBA16 -130(1D) BTME-SBA16 -130(2D) BTEB-SBA1680(1D) BTEB-SBA 16100(1D) (B)
32.7 41.0
1 day
3 days
8 days
13 days
14 days
20 days
free DAAO (unit)
100
91.0
90.7
89.1
89.0
80.1
23.3
-
14.8
92.9
89.6
activity 9.4 times higher than that of the pure silica TEOSSBA16(VN) supports and 7.5 times higher than that of the TEOS-SBA16-100(2D) (Figure 6). The most important 10909
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Table 4. Immobilized GL-7-ACA Acylase Immobilized on Mesoporous Silicas TEOS-SBA-16 and Organosilicas PMOs-SBA-16 materials
enzyme protein loading (mg/g)
enzyme protein loading yield (%)
GL-7-ACA acylase specific activity (U/ mg)a
GL-7-ACA acylase activity (U/g)b
42.3 31.3 50.6 51.1
8.8 5.8 8.3 8.4
0.22 0.58 0.56 0.26
9.1 18.2 28.3 13.5
12.7 51.9
2.1 9.5
2.03 0.43
25.9 22.4
TEOS-SBA16-100(1D) TEOS-SBA16-100(2D) BTME-SBA16-80(1D) BTME-SBA16-100(1D)ex96 BTME-SBA16-100(2D) BTEB-SBA16-80(1D) a
Enzyme activity per 1 mg of protein. bEnzyme activity per 1 g (dry weight) of catalyst; free GL-7-ACA initial activity 5.68 U/mL and initial protein 1.88 mg/mL.
The activity evolutions with reaction time of immobilized GL-7-ACA acylase stored in phosphate buffer solution (pH 8) at room temperature are listed in Table 5A. The most stable two samples are BTME-SBA-16-80(1D) and BTME-SBA-16100(2D) which retained, respectively, 52% and 43% of their initial activity after four days, compared to 80% for the free GL7-ACA acylase (Table 5B). These two samples are also the two most active samples (Figure 8 and Table 4). In the case of BTEB-SBA16-80(1D), the less ordered mesoporous structure caused by the large and flexible organic bridging group BTEB in the framework may account for the larger loss in activity compared to BTME-SBA16-80(1D). In general, enzymes (DAAO and GL-7-ACA acylase) immobilized on PMOs (BTME and BTEB) have higher activity than enzymes immobilized on siliceous material such as SBA-16 (Figures 7 and 8). As discussed above, however, the higher enzyme loadings did not correlate with higher enzyme activities in agreement with earlier reports.51,52
Table 5. (A) Stability of GL-7-ACA Acylase on Different Supports, Mesoporous Silicas TEOS-SBA-16, and Organosilicas PMOs-SBA-16 and (B) Relative Activity Variation of GL-7-ACA % (A)
variation of enzymatic activity with time (U/g at dry weight)
supports
1 day
2 days
4 days
TEOS-SBA16-100(1D) TEOS-SBA16-100(2D) BTME-SBA16-80(1D) BTME-SBA16-100(1D)-ex96 BTME-SBA16.100.2D BTEB-SBA16-80(1D) (B)
09.11 18.24 28.30 13.50 25.89 22.40 1 day
07.37 09.52 20.46 04.55 19.45 07.10 2 days
03.62 05.73 14.69 00.00 11.26 05.86 4 days
free GL-7-ACA acylase (unit)
100
92.2
80.1
sample BTME-SBA-16-80(1D) with a four times higher enzyme loading (see Figure 8). The DAAO immobilized on PMO-SBA-16 and TEOS-SBA16 supports generally displayed a higher enzymatic loading than that of GL-7-ACA acylase immobilized on the same supports. This might be explained by the difference in molecular weight and size of these enzymes.49,50 Molecular weight (72 000 Da) of GL-7-ACA acylase and size are higher than those of DAAO (39000 Da). This result underlines the need for an optimized support cage size for each enzyme investigated.
5. CONCLUSION Optimal enzyme activity and stability in reaction conditions require several precise characteristics of the support. Among these, the tridimensional cage-like structure of SBA-16 was shown to be critical for the immobilized DAAO and GL-7-ACA acylase enzyme to yield high specific activities. Moreover, the chemical nature of the cage walls is also quite significant, and the partially organic nature of the PMOs (with pore sizes
Figure 7. Activity of immobilized DAAO on different supports, silicas TEOS-SBA-16, and organosilicas PMOs-SBA-16. 10910
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Figure 8. Activity of immobilized GL-7-ACA acylase on different supports, silicas TEOS-SBA-16, and organosilicas PMOs-SBA-16. (8) Lee, C.-H.; Lin, T.-S.; Mou, C.-Y. Nano Today 2009, 4 (Issue 2), 165−179. (9) Serra, E.; Mayoral, A.; Sakamoto, Y.; Blanco, R. M.; Diaz, I. Microporous Mesoporous Mater. 2008, 114, 201−213. Serra, E.; Díez, E.; Díaz, I.; Blanco, R. M. Microporous Mesoporous Mater. 2010, 132, 487−493. (10) Chong, A. S. M.; Zhao, X. S. Catal. Today. 2004, 93−95, 293− 299. (11) Boller, T.; Meier, C; Menzler, S. Org. Process Res. Dev. 2002, 6 (4), 509−519. (12) Cao, L. Q.; Van Langen, L.; Sheldon, R. A. Curr. Opin. Biotechnol. 2003, 14 (4), 387−394. (13) Deere, J.; Magner, E.; Wall, J. G.; Hodnett, B. K. J. Phys. Chem. B 2002, 106, 7340−7347. (14) Deere, J.; Magner, E.; Wall, J. G.; Hodnett, B. K. Catal. Lett. 2003, 85, 19−23. (15) Deere, J.; Serantoni, M.; Edler, K. J.; Wall, J. G.; Hodnett, B. K.; Magner, E. Langmuir 2004, 20, 532−536. (16) Vandenberg, E. T.; Brown, R. S.; Krull, U. J. In Immobilized Biosystems in Theory and Practical Applications. Elsevier: Holland, 1983; Vol. 129. (17) Doretti, L.; Ferrara, D.; Lora, S. Biotechnology 1993, 41, 157. (18) Pollegioni, L.; Molla, G.; Sacchi, S.; Rosini, E.; Verga, R.; Pilone, M. S. Properties and applications of microbial D. amino acid oxidase: Current State and Perspectives. Appl. Microbiol. Biotechnol. 2008, 78, 1−16. (19) Huber, F.; Chauvetee, R.; Jackson, B. Preparative methods for 7aminocephalosporanic acid and 6-aminopenicillanic acid. In Cephalosporins and penicillins, chemistry and biology; Flynn, E., Ed.; Academic Press: New York, 1972; pp 27−48. (20) Inagaki, S.; Guan, S.; Fukushima, Y.; Ohsuna, T.; Terasaki, O. J. Am. Chem. Soc. 1999, 121, 9611. (21) Melde, B. J.; Holland, B. T.; Blandford, C. F.; Stein, A. Chem. Mater. 1999, 11, 3302. (22) Asefa, T.; MacLachlan, M. J.; Coombos, N.; Ozin, G. A. Nature 1999, 402, 867. (23) Olkhovyk, O.; Jaroniec, M. J. Am. Chem. Soc. 2005, 127, 60. (24) Grudzien, R. M.; Grabicka, B. E.; Pikus, S.; Jaroniec, M. Chem. Mater. 2006, 18, 1722. (25) Kleitz, F.; Solovyov, L. A.; Anilkumar, G. M.; Choi, S. H.; Ryoo, R. Chem. Commun. 2004, 1536. (26) Kleitz, F.; Kim, T. −W.; Ryoo, R. Langmuir 2006, 22, 440. (27) Gobin, O. C.; Wan, Y.; Zhao, D.; Kleitz, F.; Kaliaguine, S. J. Phys. Chem. C 2007, 111, 3053. Gobin, O. C.; Huang, Q.; Vinh-Thang, H.g.; Kleitz, F.; Eic, M.; Kaliaguine, S. J. Phys. Chem. C 2007, 111, 3059. (28) Nohair, B.; MacQuarrie, S.; Crudden, C. M.; Kaliaguine, S. J. Phys. Chem. C 2008, 112, 6065−6072.
between 8 and 10 nm) was shown to be beneficial compared to silica SBA-16. DAAO and GL-7-ACA acylase enzyme adsorption and activity experiments indicate that the framework composition of the mesoporous organosilicas has a great influence on the enzyme adsorption and activity behavior of these materials. In conclusion, the periodic mesoporous organosilica PMO-SBA16 supports are suitable for immobilizing DAAO and GL-7-ACA acylase enzymes. The hydrothermal temperature during PMO synthesis which affects both cage and cage aperture diameters, was shown to be a critical parameter for enzyme retention and specific activity. It is expected that PMO materials with large cavity and pore aperture sizes in combination with hydrophobic enough surface area can be used for the adsorption of numerous other enzymes.
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ASSOCIATED CONTENT
S Supporting Information *
Figures 1S−3S. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (S.K.); E-mail:
[email protected] (P.Q.T.) Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was partially funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.03-2011.07 and by the Natural Science and Engineering Council of Canada.
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