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Stability and Catalytic Kinetics of Horseradish Peroxidase Confined in Nanoporous SBA-15 Hideki Ikemoto, Qijin Chi,* and Jens Ulstrup* Department of Chemistry and NanoDTU, Technical UniVersity of Denmark, DK-2800 Lyngby, Denmark ReceiVed: April 7, 2010; ReVised Manuscript ReceiVed: August 20, 2010
We have synthesized nanoporous silica, SBA-15, in the 1 µm size range with the pore diameter of 7.6 nm. The redox enzyme horseradish peroxidase (HRP) was entrapped in the pores to form nanostructured hybrid materials. The catalytic activity of free and immobilized enzyme was first compared at room temperature. Details of the enzyme kinetics including the apparent Michaelis constant (KM) and maximum rate (Vmax) were determined. Both thermal stability and stability, toward the denaturing agents guanidinium chloride and urea, of free and immobilized enzymes were compared next. The thermal stability of the immobilized enzyme is significantly improved in comparison with free HRP. The catalytic kinetics is slowed down notably, but Vmax is much more robust to heat than the free enzyme. The stability resistance of the enzyme toward the denaturing agents depends on the chemical nature of the denaturing agents and interactions between enzyme and silica nanopore walls. Guanidinium chloride showed similar attenuation of the catalytic activity of immobilized and free enzyme. In contrast, the immobilized HRP was much more resistant to urea than the free enzyme. The different behavior of free and immobilized enzyme is most likely due to different hydrogen bonding of water and increased hydration strength of the protein inside the nanopores. 1. Introduction The confinement of biomacromolecules in a nanoscale environment provided by solid host materials is of significant interest in biotechnology such as biocatalysis, biosensors, and drug delivery, as the hybrid combination extends applications of biological molecules well beyond their natural conditions. Several types of inorganic host materials have been developed, among which silica-based nanoporous materials offer significant advantages because of their ordered structure and uniform pore size.1 The first synthesis of mesoporous molecular sieves known as MCM-41 was reported in 1992.2 Different types of nanoporous silica have since been synthesized,3 for example, Santa Barbara Amorphous-15 (SBA-15)4 and mesocellular silica foam (MCF).5 Mesoporous silica materials are attractive hosts for encapsulation of biological macromolecules because they offer controlled pore size, high surface area, and large pore volume. In addition, the inside nanopore walls can be functionalized using surface chemistry of silica materials, offering new routes, for example, to surface chemical properties of the nanopores and new bioimmobilization methods. Immobilization or adsorption studies of proteins in mesoporous silica have been broadly reported.6-10 The adsorbed amount of proteins depends on the pore volume and the composition of the adsorbent7 as well as on particle size and morphology of the adsorbent.8 Many attempts to encapsulate proteins and enzymes in mesoporous silica for potential applications in biocatalysis,11,12 drug delivery,13,14 and biosensors have also been reported.15 These strategies have recently been extended to multiple proteins confined simultaneously in the nanopores.16 Understanding of stability and catalytic activity of proteins or enzymes confined in nanoporous materials is the basis for their applications. In addition, many natural biological reactions take place on the mesoscopic scale in “crowded” or confined * To whom correspondence should be addressed. (Q.C.) Tel: +45 4525 2032. Fax: +45 4588 3136. E-mail:
[email protected]. (J.U.) Tel: +45 4525 2359. Fax: +45 4588 3136. E-mail:
[email protected].
space. Studies of the catalytic activity and stability of enzymes in different nanoscale environments or inside mesoporous materials therefore offer clues to fundamental understanding of these aspects of biological reactions. As an example, Itoh et al. showed that several proteins immobilized in mesoporous silica display increased resistance to organic solvents,17 and Zhao et al. showed that immobilized papain exhibits much better pH stability.18 Denaturing agents are effective probes to elucidate the stability of proteins confined in nanoscale space. Investigations of the resistance to denaturing agents has been extended to proteins and enzymes inside mesoporous silica.19,20 Hemoglobin encapsulation resulted, for example, in significant improvement in retaining biological activity against guanidinium chloride (GdmCl).20 Lei et al. observed synergistic effects of functionalized mesoporous silica and the denaturing agent urea on the enzyme activity of glucose isomerase encapsulated in mesoporous silica.19 Takahashi et al. reported direct relations between the catalytic activity of the heme enzyme horseradish peroxidase (HRP) immobilized inside mesoporous silica and the surface properties and size of the nanopores.21 In the present work, we have studied the stability and the catalytic kinetics of HRP22 encapsulated into mesoporous silica SBA-15 as host nanoporous material. SBA-15 can be synthesized by controlled routes to have thick walls and hexagonally aligned ordered pores (5-30 nm) that allow immobilization of biological macromolecules inside the pores. HRP catalyzes the oxidation of aromatic compounds by H2O2. Takahashi et al. investigated the catalytic activity of HRP immobilized inside the pores and found that both surface character and pore size are crucial to achieve high catalytic activity.21 In another study by Chouyyok et al.,23 three types of mesoporous silica (MCM41, SBA-15, and MCF) were used, and the effects of pH on HRP immobilization were investigated. The largest amount of enzyme was loaded in MCF, and maximum catalytic activity was obtained at pH 6.
10.1021/jp103137e 2010 American Chemical Society Published on Web 09/10/2010
Stability and Catalytic Kinetics of HRP The kinetics and catalytic mechanism of HRP immobilized inside SBA-15 pores have been investigated in greater detail in the present report. Synthesized SBA-15 with pore diameter of 7.6 nm was used as the host material, suitable for confinement of HRP molecules by physical or electrostatic entrapment. In particular, the enzyme kinetic constants were obtained by different methods and compared with those of the free enzyme. Both free and confined HRP show high catalytic activity toward oxidation of aromatic compounds by H2O2 (phenol/4-aminopyrine/H2O2 assay24) but with notably different catalytic kinetics. Thermal stability and resistance to the denaturing agents guanidium chloride and urea were also investigated. Intriguing differences not reported before and quantified by the kinetic analysis of free and pore-confined HRP catalysis were found. Views on their different physical origin are also discussed. 2. Experimental Section 2.1. Chemicals. Horseradish peroxidase P8250-5KU Type II (EC number: 1.11.1.7, 150-250 units/mg solid, SigmaAldrich), urea (minimum, 99.5%), tetraethyl orthosilicate (98% (GC), Aldrich), poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (denoted as Pluronic P-123, Sigma-Aldrich), 4-aminoantipyrine (g98.0%, Sigma-Aldrich), phenol (g99.5%, Sigma-Aldrich), and 30% hydrogen peroxide (Fluka) were used as received. The precise H2O2 concentration was determined by MnO4- titration. Other agents were at least of analytical grade. Milli-Q water (18.2 MΩ cm) was used throughout. 2.2. Synthesis. SBA-15 was synthesized by a slightly modified reported procedure.25 In brief, Pluronic 123 (4.0 g) was added to 144 mL of 1.7 M HCl, and the mixture was stirred at 40 °C for 4 h. Tetraethyl orthosilicate (8.01 g) was then added dropwise, and the mixture was further stirred for 2 h. The resulting gel was incubated at 100 °C for 24 h. The final product was filtered, washed with water three times, and dried at 80 °C overnight. When required, the synthesized material was further calcined at 500 °C for 6 h. 2.3. Characterization. X-ray diffraction (XRD) patterns of the nanoporous silica material were collected on a PANalytical’s X’Pert PRO X-ray diffractometer using Cu KR as radiation. The diffractograms were recorded in the 2θ range of 0.6 to 5°. Nitrogen isotherms were measured at 77 K using a Micromeritics ASAP 2020 system. The calcined SBA-15 samples were degassed at 200 °C for 2 h. The specific surface area of the sample was calculated using the Brunauer-Emmett-Teller (BET) method.26a The pore size distribution was determined from the desorption isotherm measurements using the Barrett-Joyner-Halenda (BJH) method.26b A Helios EBS3 instrument was used to record SEM images. Silica particles for SEM were coated with gold to 3.0 nm thickness by sputtering. TEM images were obtained using a Tecnai T20 instrument operated at 200 kV using a CCD camera (Gatan). To prepare TEM specimens, a lacey carbon grid was put into the powder a few times. This ensured that the particles would stick to the grid. 2.4. Immobilization of HRP in SBA-15. HRP solution (2.5 mL, 0.5 mg mL-1) was added to 125 mg SBA-15 placed in a centrifuge tube. The mixture was stirred at 4 °C for 48 h. The loading of HRP into the nanopores was monitored by UV-vis spectra at 409 nm after centrifugation using a UV-vis spectrophotometer (HP8453, Hewlett Packard) until no apparent decrease in absorbance was detected in the supernatant. The solution was then centrifuged and washed several times with phosphate buffer (pH 7.4) until no free enzyme in the supernatant could be detected. From absorbance changes, the
J. Phys. Chem. C, Vol. 114, No. 39, 2010 16175 total amount of HRP immobilized was estimated to be ca. 7.1 mg per gram SBA-15. The HRP-SBA-15 conjugated material (in solid-state form) was dried and stored at 4 °C. 2.5. Assays of Enzyme Activity. The catalytic activity of free and immobilized enzyme was determined using a modified reported colorimetric procedure.24 For the free enzyme, a solution containing 2.4 mM 4-aminoantipyrine (4-AAP), 86 mM phenol, 0.077 to 1.5 mM hydrogen peroxide, and 25 mM phosphate buffer (pH 7.4) in a total volume of 1.21 mL was prepared in a cuvette, and 40 µL of 0.01 mg mL-1 HRP was added and mixed. The absorbance at 510 nm was monitored to determine the initial reaction rate. For the immobilized enzyme, 13.6 mg of HRP-containing SBA-15 was mixed with 12.5 mL of solution of 2.4 mM 4-AAP, 86 mM phenol, 0.077 to 1.5 mM hydrogen peroxide, and 25 mM phosphate buffer (pH 7.4). The samples (1 mL) were centrifuged at given intervals to remove the particles, and the corresponding absorbance of the supernatant at 510 nm was recorded. The extinction coefficient used for calculation is 7100 M-1 cm-1. Full time records from which kinetic constants at given substrate concentrations could be obtained were also analyzed. 2.6. Thermal Stability. We examined the thermal stability of HRP immobilized in SBA-15 by adding 13.6 mg HRPimmobilized SBA-15 to 2.2 mL of 100 mM phosphate buffer (pH 7.4) and heating the mixture for 30 min at 70 °C. The solution was left to cool to room temperature, and the catalytic activity was determined. The free enzyme, 40 µL of HRP (0.01 mg mL-1), was treated similarly. 2.7. Stability in the Presence of Denaturing Agents. Free or immobilized HRP was incubated for 22-28 h in solutions of varying denaturant concentrations. Catalytic activity in the denaturant solutions was determined after incubation, as described above. 3. Results 3.1. Synthesis and Immobilization. SBA-15 synthesized by the slightly modified reported procedure15 was characterized by SEM, TEM, and XRD. The SBA-15 particles were rod-shaped. The average particle size is several hundred nanometers in diameter and 1 µm in length (Figures S2 and S3 in the Supporting Information), with hexagonally ordered structures (Figures S1 and S3 in the Supporting Information). The pore diameter of calcined SBA-15 is 7.6 nm (Figure S4 in the Supporting Information), suitable to accommodate HRP (∼3.7 nm ×4.3 nm ×6.4 nm21). Pore surface area and pore volume determined from dinitrogen adsorption experiments were 872 m2 g-1 and 1.3 cm3 g-1, respectively. Immobilization of HRP was achieved by suspending nanoporous material in HPR solution (pH 7.4). Because the isoelectric point of HRP is 8.9, HRP molecules are thus positively charged in the solution used. The silanol groups inside the nanopores are deprotonated at pH 7.4. Electrostatic interactions between HRP molecules and the inner nanopore wall are thus important if not dominating in enzyme immobilization. 3.2. Catalytic Activity at Room Temperature. Catalytic activity of the immobilized HRP and the effect of the H2O2 concentration were first investigated. The initial velocity was determined from the absorbance change at 510 nm. Figure 1 shows an example of absorbance-time relations at a H2O2 concentration of 1.2 mM. The exact amount of accessible active enzyme immobilized could not be determined. Direct clues to the accessibility issue and to the amount of pore-confined enzyme that retains, albeit reduced, enzyme activity are in fact quite elusive, but irreversibly bound inhibitors may offer a way. We shall discuss this issue further in Section 4. However, the
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Figure 1. Time courses of product (N-antipyryl-p-benzoquinoeimine, APBQ) formation in the oxidation of phenol by H2O2. Free or immobilized enzyme was mixed with a solution containing 2.4 mM 4-AAP, 86 mM phenol, 1.2 mM hydrogen peroxide and 25 mM phosphate buffer (pH 7.4). Filled circles and triangles show the response from free and immobilized enzyme, respectively.
concentration of accessible HRP in the immobilized state is expected to be significantly higher than that of free HRP in solution because the amount of enzyme used in the activity assays is 7.7 and 0.38 µg mL-1 for the immobilized and free state, respectively. Even so, the initial rate of product formation is much lower for the immobilized than for the free enzyme. Separate adsorption experiments did not detect any adsorbed product. The Michaelis constant KM and the maximum rate constant Vmax were determined from Lineweaver-Burk or directly from Michaelis-Menten plots (Figures S5 and S6 in the Supporting Information). The use of these frames in their simplest form raises questions regarding the mechanism of the assay reaction because the reaction is certainly more complicated than inherent in straightforward use of Lineweaver-Burk or MichaelisMenten analysis and involves in fact two substrate molecules.24 Lineweaver-Burk or Michaelis-Menten analysis or equivalent analysis directly of the time evolution of the process at each given substrate concentration, however, still offers applicable operational frames with reference to the dominating assay process
PhOH + Am-NH2 + 2H2O2 f APBQ + 4H2O
(1) The main reservation is then that the catalytic constants KM and Vmax are composite quantities and acquire the status of “apparent”. The “apparent” KM values of free and immobilized HRP from the former plots were 0.47 ( 0.06 and 0.35 ( 0.03 mM, respectively, and the apparent Vmax values were 160 ( 20 and 9.3 ( 0.7 µM min-1. Similar values were obtained by the latter method (using an Excel solver program), giving Vmax of free and immobilized HRP of about 120 ( 10 and 8.6 ( 0.2 µM min-1, respectively, and KM ) 0.27 ( 0.03 and 0.31 ( 0.01 mM. Table 1 summarizes the catalytic constants obtained. Vmax of immobilized HRP is significantly lower than that of free HRP. Several reasons for this can be offered. The active site of some of the enzyme adsorbed in the SBA-15 pores may not be accessible. The conformation of some of the enzyme may also have been changed by electrostatic interaction with the silica surface. Diffusion constraints are other possible reasons. Recently, Jaladi et al. investigated the effects of diffusion resistance on the activity of an immobilized enzyme (Burkhold-
eria cepacia lipase).28 The enzyme immobilized in SBA-15 with 5.5 nm pores, comparable to the pore diameter in the present study (7.6 nm), showed only 20-30% of catalytic activity for free lipase. The enzyme was, however, more catalytically active in SBA-15 with large pore diameter (24 nm), most likely because of fewer diffusion limitations.28 TEM further revealed that some particles were connected end-to-end, which would make it even more difficult for substrates to reach the enzyme or for the products to escape from the pores to the outside. 3.3. Thermal Stability. The stability of immobilized HRP to heat was investigated next. After incubation at 70 °C for 30 min, the catalytic activity was determined after cooling to room temperature. The initial rate of product formation has decreased to 73% after the free enzyme had been incubated at 70 °C, indicating that the enzyme has denatured extensively (Figure 2). In contrast, the initial rates of product formation catalyzed by immobilized enzyme are about the same before and after incubation at 70 °C (Figure 3). These results indicate that immobilized enzyme improves the thermal stability significantly, holding promise for exploiting the enzyme at elevated temperatures where the free enzyme is too unstable. 3.4. Stability in the Presence of Denaturing Agents. The stability of the immobilized enzyme against guanidinium chloride (GdmCl) and urea was finally investigated. Immobilized enzyme behaved in much the same way on GdmCl addition as the free enzyme (Figure 4). Significant activity decrease is noted at 1 to 2 M GdmCl, followed by further strong decrease at [GdmCl] > 4 M. The plateau suggests unfolding in a two-step process. In contrast, immobilized HRP incubated with urea showed strongly improved resistance to denaturation (Figure 5). The immobilized enzyme has retained 80% activity, even at 5 M urea, where the activity of the free enzyme has dropped to
