Langmuir 2005, 21, 11099-11104
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Immobilization of Myoglobin on Phosphate and Phosphonate Grafted-Zirconia Nanoparticles Francesca Bellezza, Antonio Cipiciani,* and Maria Anna Quotadamo Dipartimento di Chimica, Universita` di Perugia, via Elce di Sotto, 8, 06123 Perugia, Italy and “Centro di Eccellenza Materiali Innovativi Nanostrutturati” (CEMIN), Universita` di Perugia, Via Elce di Sotto 10, 06123 Perugia, Italy Received June 6, 2005. In Final Form: September 6, 2005 We investigated the adsorption and catalytic activity of myoglobin (Mb) immobilized on colloidal particles of zirconia covalently grafted with phosphoric (ZrO2-P) and benzenephosphonic acid (ZrO2-BP). The maximum adsorption was reached after 1 h of contact and was greater on a hydrophilic support, ZrO2-P, compared to a hydrophobic support, ZrO2-BP. The equilibrium isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the surface of the nanoparticles. The nanostructured biocomposites are active in the oxidation of 2-methoxyphenol (guaiacol) by hydrogen peroxide. The oxidation catalyzed by immobilized Mb followed a Michaelis-Menten kinetics, similar to that observed in the oxidation by free Mb. Furthermore, the catalytic efficiency is similar to that of free Mb and higher than that of “large-size” biocatalysts (with sizes larger than 1 µm). In the latter case, the kinetic parameters, kcat and KM, indicate that this is mostly due to an increased affinity of the nano-biocomposite for the substrate. The activity of the nano-biocomposites decreases slightly as the amount of adsorbed protein increases. This is mainly due to the formation of a nonordered monolayer, which reduces the accessibility of the substrate to the active center.
Introduction Enzymes are biological catalysts with excellent activity and stereoselectivity that can also be used to catalyze chemical transformations but, in this latter case, their use is severely limited by poor stability and chemical sensitivity. To overcome these limitations, several strategies have been applied including the immobilization of biomaterial on insoluble supports. Enzyme immobilization not only improves their stability under extreme conditions but also allows them to be easily separated from the reaction mixture and reused. Natural organic polymers, synthetic organic polymers, and inorganic materials have been used as carriers.1-3 Inorganic matrixes have good mechanical properties and thermal stability and are resistant to microbial attack and organic solvents.4,5 However, compared with organic materials, most of the inorganic supports have a smaller external surface and a lack of reactive functional groups.6 Recently, we used zirconium phosphate R-Zr(HPO4)2 (R-ZrP) and zirconium phosphonates such as R-Zr(C6H5PO3)2 (R-ZrBP) and R-Zr(HOOCCH2CH2PO3)2 (R-ZrCEP) for the immobilization of myoglobin (Mb)7 and lipase from Candida rugosa (CRL).8,9 These layered inorganic solids * Corresponding author. E-mail:
[email protected]. Telephone: 075/585540. Fax: 075/5855560. (1) Miksa, B.; Slmokowski, S. Colloid Polym. Sci. 1995, 273, 47. (2) Hellsten, M. Colloid Polym. Sci. 1992, 270, 1188. (3) Yoshinaga, K.; Kito, T.; Yamaye, M. J. Appl. Polym. Sci. 1990, 41, 1443. (4) Reetz, M. T.; Zonta, A.; Simpelkamp, J. Biotechnol. Bioeng. 1996, 49, 527. (5) Gao, L.; Bornscheuer; Schmid, R. D. J. Mol. Catal. B: Enzym. 1999, 6, 278. (6) Nguyen, D.; Smit, M.; Dunn, B.; Zink, J. L. Chem. Mater. 2002, 14, 4330. (7) Bellezza, F.; Cipiciani, A.; Costantino, U.; Nicolis, S. Langmuir 2004, 20, 5019. (8) Bellezza, F.; Cipiciani, A.; Costantino, U.; Negozio, M. E. Langmuir 2002, 18, 8737. (9) Bellezza, F.; Cipiciani, A.; Costantino, U. J. Mol: Catal. B: Enzym. 2003, 26, 47.
have many positive features including an ordered structure, different surface areas, and well-defined hydrophilic/ hydrophobic surface characteristics which make them attractive candidates for the immobilization of bioactive materials. In the case of CRL, we obtained a selective immobilization of CRL isoenzymes that allowed the preparation of some biocomposites with different catalytic properties.9 The adsorption of myoglobin onto zirconium phosphonates allowed heterogeneous biocatalysts to be obtained that had good catalytic efficiency, had increased resistance toward inactivation by hydrogen peroxide, and could be stored for months without a significant loss of catalytic activity.7 Continuing our research on the immobilization of enzymes and proteins onto solid supports, organicinorganic hybrid nanocomposites based on zirconium oxide have been considered. These materials can be synthesized by grafting organic functions onto the surface of inorganic particles through strong chemical bonds. Due to the stable zirconium-phosphate bond, the zirconium oxide surface can be modified with different phosphates and phosphonates that provide the external surface of supports with specific functions that can interact with biomolecules.10-14 Figure 1 shows a schematic representation of the processes involved in assembling these new biocomposites. The support core is made of monoclinic zirconia particles of size lower than 100 nm obtained by hydrothermal treatment of zirconium acetate.14 The advantages of using nanoparticles as supports for the immobilization of enzymes and proteins are (i) a high specific surface area per unit mass for binding large amounts of biomaterial, and (ii) a low mass transfer (10) Schafer, W. A.; Carr, P. W. J. Chromatogr. 1991, 587, 137. (11) Randon, J.; Blanc, P.; Paterson, R. J. Membr. Sci. 1995, 98, 119. (12) Gao, W.; Dickinson, L.; Grozinger, C.; Morin, F.; Reven, L. Langmuir 1996, 12, 6429. (13) Clausen, A.; Car, P. Anal. Chem. 1998, 70, 378. (14) Carrie`re, D.; Moreau, M.; Barboux, P.; Boiolot, J. P.; Spalla, O. Langmuir 2004, 20, 3449.
10.1021/la051487y CCC: $30.25 © 2005 American Chemical Society Published on Web 10/14/2005
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Experimental Section
Figure 1. Schematic representation of processes involved in the nanobiocomposite assembly and its use in biocatalysis.
resistance due to the Brownian motion of the nanobiocomposites. Up to now, only a few studies have been devoted to a quantitative analysis of the activity of enzymes and proteins bound to colloidal particles.15-19 The data obtained by applying the classical Michaelis-Menten kinetics seems to be appropriate for a quantitative comparison between bound and free biocatalysts. In this way, the results obtained with enzymes and proteins adsorbed onto colloidal particles can be compared with those obtained with other methods of immobilization. The objectives of the present study were to immobilize Mb on a new type of colloidal particles characterized by different hydrophobic/hydrophilic groups on the surface and to analyze the effect of particle dimensions and of surface characteristics on the properties of the obtained biocatalyst. Mb was chosen as a representative example for investigating the peroxidase-like activity of a supported protein because of its availability, well-known structure, small size, and strong spectroscopic signatures that allow protein binding and redox activity to be monitored. In addition, detailed data on the peroxidase activity of several Mb derivatives have recently been reported in the literature.20-23 Oxidative dehydrogenation is the classical reaction catalyzed by peroxidases. In this reaction, hydrogen peroxide reacts rapidly with Mb to generate an intermediate (known as compound I) which is capable of oxidizing amines, phenols, and other organic compounds.24 The mechanism of these reactions generally proceeds through one-electron steps. In fact, one-electron reduction of compound I produces a second protein intermediate, compound II, which can also monoelectronically oxidize a molecule of substrate, albeit at a slower rate. (15) Jin, W.; Shi, X.; Caruso, F. J. Am. Chem. Soc. 2001, 123, 8121. (16) Oh, J.-T.; Kim, J.-H. Enzyme Microb. Technol. 2000, 27, 356. (17) Caruso, F.; Trau, D.; Mohwald, D.; Renneberg, R. Langmuir 2000, 16, 1485. (18) Caruso, F.; Schuler, C. Langmuir 2000, 16, 9595. (19) Lvov, Y.; Caruso, F. Anal. Chem. 2001, 73, 4212. (20) Dawson, J. H. Science 1988, 240, 433. (21) Matsui, T.; Ozaki, S.; Liong, E.; Phillips, G. N., Jr.; Watanabe, Y. J. Biol. Chem. 1999, 274, 2838. (22) Monzani, E.; Alzuet, G.; Casella, L.; Redaelli, C.; Bassani, C.; Sanangelantoni, A. M.; Gullotti, M.; De Gioia, L.; Santagostini, L.; Chillemi, F. Biochemistry 2000, 39, 9571. (23) Ozaki, S.; Roach, M. P.; Matsui, T.; Watanabe, Y. Acc. Chem. Res. 2001, 34, 818. (24) Redaelli, C.; Monzani, E.; Santagostini, L.; Casella, L.; Sanangelantoni, A. M.; Pierattelli, R.; Banci, L. ChemBioChem 2002, 3, 226.
1. Materials. Myoglobin (from horse heart, 90% pure, LOT 122K7057) was obtained from Sigma and purified by an ionexchange chromatography on a Sephadex G-50 column. 2. Hydrothermal Synthesis of Colloidal Zirconia. Colloidal solution of zirconia was prepared following a method described in ref 14. A 9 mL volume of zirconium acetate (16% Zr in 2-propanol, Aldrich) was placed in an autoclave and treated at 210 °C for 2 h. The colloidal solution was washed by successive centrifugation steps (12000 rpm, 15 min) and redispersed in distilled water until no residual zirconium ions could be found in the solution. The solid was collected after drying the colloidal solution under vacuum at 60 °C and characterized by X-ray powder diffraction (XRD), thermogravimetric analysis (TG-DTA), and B.E.T. surface area. 3. Covalent Grafting of Zirconia Colloid with Phosphate and Phosphonates. Grafted zirconia nanoparticles were prepared by a process described in ref 14. The colloidal aqueous solution of zirconia (100 mL, 0.12M) was added to an aqueous solution of phosphoric or benzenephosphonic acid (100 mL, 0.06 M). The resulting mixture ([zirconia] ) 0.06 M, P/Zr ) 0.5) was heated at 110 °C for 4 h. Any excess of acid was then washed out by centrifugation and dispersions in water. The colloids, ZrO2-P and ZrO2-BP respectively, were dried under vacuum at 60 °C and then characterized by XRD, TG-DTA, and BET surface area. 4. Characterization of Resulting Nanoparticles. The morphology of the particles was studied using a Philips 208 transmission electron microscope. A small drop of colloidal solution was deposited on a copper grid precoated with a film of Formwar and then evaporated in air at room temperature. Phosphate and benzenephosphonate analysis was performed by ion chromatography using a Dionex 500 instrument with a Dionex AS4A column. The X-ray powder diffraction (XRD) patterns were taken with a computerized Philips PW1710 diffractometer using the Cu KR radiation, operating at 40 kV and 20 mA, step scan 1° min-1. The specific surface areas were calculated according to the BET method from N2 adsorption isotherms at 77K taken with a computer controlled Micromeritics ASAP2010 instrument. Thermogravimetric analysis (TG-DTA) was performed with a Netzsch STA 449C thermobalance. 5. Immobilization of Mb on Grafted-Zirconia Nanoparticles. The colloidal solution of grafted-zirconia was characterized in terms of concentration by drying a fixed volume and weighting the resulting solid. A 4% of ethanol was added to the ZrO2-BP solution to improve the wettability. The pH of the colloidal solution was then measured (3.4) and a solution of NaOH 0.2 M was added until pH 7.0. A volume of colloidal solution, containing about 10 mg of solid, was added to an aqueous solution of Mb at different concentrations. The mixture was stirred for 1 h at room temperature and the immobilized protein was recovered by centrifuging at 11000 rpm for 15 min. The supernatant was assayed for protein concentration by UV spectra (λ ) 410 nm, ) 160 000 M-1 cm-1). The amount of bound protein was calculated from the difference between the concentration of protein in the initial solution and in the supernatant. The pellet was washed and resuspended in water, obtaining a suspension with a known concentration of immobilized Mb to use for catalysis. 6. Enzyme Kinetics for Immobilized Protein. The peroxidase activity of immobilized protein were evaluated by measuring the initial oxidation rates of 2-methoxyphenol by hydrogen peroxide. A fixed volume of biocomposite suspension was added in a cuvette containing the substrate to obtain a final volume of 3 mL (0.2 M phosphate buffer, pH 6.0). The kinetics were started by adding hydrogen peroxide. The kinetic constants kcat and KM were obtained as described in ref 7.
Results and Discussion 1. Characterization of the Particles. The hydrothermal treatment of the zirconium acetate solution yields a colloidal suspension of zirconium oxide nanoparticles.
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Figure 4. Thermogravimetric analysis (TG-DTA) of ZrO2-P particles.
Figure 2. TEM micrographs of (a) zirconia particles, (b) zirconia particles grafted with phosphoric acid, (c) zirconia particles grafted with benzenephosphonic acid, and (d) myoglobin adsorbed onto zirconia particles grafted with benzenephosphonic acid.
Figure 5. Thermogravimetric analysis (TG-DTA) of ZrO2BP particles.
Figure 3. Thermogravimetric analysis (TG-DTA) of zirconia nanoparticles (ZrO2-Ac).
TEM micrographs show a system of well dispersed particles with diameters less than 100 nm (Figure 2). The X-ray diffraction pattern of the solid, collected after drying, is characteristic of monoclinic zirconia (data not shown) and the BET analysis of N2-adsorption isotherms indicates the formation of particles with 113 m2/g surface area. Thermogravimetric analysis of the solid shows a twostep weight loss: the first, that occurs at temperatures less than 200 °C, corresponds to the water loss, whereas the second, at temperatures around 300 °C, corresponds to the degradation of the acetate groups (see Figure 3). From the weight loss, a chemical composition of ZrO1.9(CH3COO)0.2‚1.7 H2O (ZrO2-Ac) can be calculated. Phosphoric and benzenephosphonic acids can be bound onto zirconia particles by heating a mixture of colloidal ZrO2 and the specific acid ([ZrO2] ) 0.06 M, [Acid] ) 0.03 M, P/Zr ) 0.5) at 110 °C for 4 h. After grafting, the particles remain discrete with a mean diameter similar to that of the starting zirconia (see Figure 2). The XRD patterns of the particles grafted with phosphoric (ZrO2-P) and benzenephosphonic acid (ZrO2-BP) are those of the initial zirconia.
The absence of a typical lamellar zirconium phosphate or benzenephosphonate pattern indicates that the grafting reaction of zirconia nanoparticles is a surface reaction and the core of the particles is not attacked. The BET surface areas of the dried particles remained large (132 m2/g for ZrO2-P and 147 m2/g for ZrO2-BP). The data plot for the thermogravimetric analyses for the grafted particles are shown in Figures 4 and 5. The following chemical compositions can be calculated: ZrO1.79(O2P(OH)2)0.42‚3.7 H2O (ZrO2-P) ZrO1.75(OP(OH)(C6H5))0.50‚0.53 H2O (ZrO2-BP) The IR spectra of the particles obtained after centrifuging and drying were identical to those reported in the literature.25 2. Adsorption of Myoglobin. The Mb adsorption onto ZrO2-P and ZrO2-BP as a function of time was examined at an initial concentration of 0.303 mg Mb tot/mg supp and the results are shown in Figure 6. The adsorption of Mb onto grafted zirconia was very fast and reached the maximum amount within 10 min. The maximum amount of Mb adsorbed onto ZrO2-P was larger than that adsorbed onto ZrO2-BP (Figure 7). The adsorption isotherms may be described by the Langmuir equation26 (1)
Qe )
QMAXCe (1/aL) + Ce
(1)
where Ce is the concentration of Mb in solution at equilibrium (mg Mb/mL), Qe is the amount of Mb adsorbed onto the support (mg Mb/mg supp), QMAX is the maximum
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Bellezza et al. Table 1. BET Surface Area, Phosphate/ Benzenephosphonate Content and Density of the Particles Obtained after Drying system
BET surface area (m2/g)
phosphorousa (mmol/g)
density (g/cm3)
ZrO2-Ac ZrO2-P ZrO2-BP
113 132 147
1.6 2.1
2.0 1.5 1.5
a
Determined by chromatographic method.
Table 2. Adsorption Parameters for Mb onto Grafted-Zirconia Particles with the Langmuir Model
Figure 6. Rate of adsorption of Mb onto ZrO2-P and ZrO2BP (0.303 mg Mb tot/mg supp).
system
aL (mL/mg Mb)
QMAX (mg Mb/mg supp)
r2
ZrO2-P ZrO2-BP
117.3 7142.8
0.205 0.157
0.95 0.92
the presence of a monolayer of protein molecules on the regular and homogeneous surface of the supports. Since the approximate dimension of Mb is 4.5 × 3.5 × 2.5 nm, 1 mg of Mb, adsorbed as a closely packed monolayer, should cover 0.30-0.52 m2, depending on whether the molecules are adsorbed side-on or end-on. Considering the maximum amount of protein adsorbed onto grafted-zirconia particles (see QMAX in Table 2), we obtained a value of saturated surface of 61.5-106.6 m2/g for ZrO2-P and 47.1-81.6 m2/g for ZrO2-BP. It is interesting to compare these calculated saturated surface areas with the surface area of the nanoparticles. To evaluate the expected order of magnitude of the specific surface area of the colloidal suspension, we considered the mean diameter of the particles to be 50 nm (approximated to spheres) and a density of 1.5 g/cm3 (like that of the dried material). This corresponds to a calculated surface area of 80 m2/g, according to the following equation (2):27
S ) 6000/d × F Figure 7. Equilibrium adsorption isotherms of Mb onto ZrO2-P and ZrO2-BP (t ) 1 h).
(2)
where S is the surface area (m2/g), d (nm) is the diameter, and F is the density (g/cm3). These approximate calculations show that the available external surface of supports is completely covered by a monolayer of Mb molecules as suggested by the fitting of the Langmuir equation. 3. Enzymatic Activity. The enzymatic activity was evaluated in terms of redox activity. The scheme of the substrate oxidation by peroxidase is as follows:28
(a) E + H2O2 h (E-H2O2) f E-I + H2O (b) E-I + SH h (E-I-SH) f E-II + S• + H+ (c) E-II + SH + H+ h (E-II-SH) f E + S• + H2O where E-I and E-II are, respectively, compounds I and II. Generally, step c is the rate-limiting step. When step b is much faster than step c, the scheme can be reduced to a simple bimolecular ping-pong mechanism. The resulting rate equation (3) is24 Figure 8. Adsorption isotherms of Mb onto ZrO2-P and ZrO2BP according to the Langmuir equation (1).
adsorption capacity of the support (mg Mb/mg supp), an aL is the Langmuir constant (mL/mg Mb). Figure 8 shows the results of treatment of experimental data according to eq 1, and the shapes obtained indicate
kcat[E0]
V) 1+
KM(H2O2) [H2O2]
+
KM(S)
(3)
[S]
where KM(S) contains terms which refer only to step c. (25) Randon, J.; Blanc, P.; Paterson, R. J. Membr. Sci. 1995, 98, 119. (26) Al-Duri, B.; Yong, Y. P. J. Mol: Catal. B: Enzym. 1997, 3, 177.
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Figure 9. Initial rate of Mb/ZrO2-BP(1) catalyzed oxidation of 2-methoxyphenol as a function of substrate concentration.
To evaluate the primary kinetic data in terms of the classical Michaelis-Menten equation, the term KM(H2O2)/ [H2O2] must be negligible. This condition is fulfilled when the hydrogen peroxide concentration is high enough to make step a faster than step c. The conditions for hydrogen peroxide saturation were set up for the substrate and the kinetic data were analyzed with the Michaelis-Menten equation
V ) Vmax [S]/KM + [S]
(4)
where Vmax ) kcat[E0] is the rate obtained at infinite concentration of substrate and kcat (turnover number) gives a measure of the activity of the protein. KM is the Michaelis constant and provides a measure of the affinity of the protein for the substrate. The oxidation of 2-methoxyphenol, catalyzed by Mb adsorbed on grafted zirconia nanoparticles, was performed. The rates of substrate oxidation by the immobilized Mb showed Michaelis-Menten saturation kinetics (Figure 9), demonstrating that the immobilized protein reacts similarly to Mb in solution. The apparent kinetic parameters determined for the experimental data using eq 4 are reported in Tables 3 and 4. It appears that the values of kcat, KM, and kcat/KM were affected by the adsorption of Mb onto the supports. The constant kcat is smaller than the value for the free protein, indicating a lower electron-transfer rate from the substrate to the enzyme. The decrease of activity upon immobilization onto large-size solid materials has been already observed,29 and it is due to a combination of several factors, such as hindrance of accessibility of substrate to the active site, multiple point binding or partial damage of the protein. In our case, the decrease of kcat was similar for all of the biocatalysts examined (Mb/ZrO2-P and Mb/ ZrO2-BP). A little difference was observed for the hydrophilic support ZrO2-P, where the decrease was smaller than that observed for the hydrophobic support ZrO2BP. This fact could be due to a different interaction between protein and supports. Although the driving forces responsible for adsorption are still not well understood, it is possible to assume that for Mb/ZrO2-P the main contribution is given by electrostatic interactions between positively charged lysine (27) Rouquerol, F.; Rouquerol, J.; Sing, K. Adsorption by Powders and Porous solids; Academic Press: London, 1999. (28) Dunford, H. B. Heme Peroxidase; Wiley-VCH: New York, 1999. (29) Kondo, A.; Fukuda, H. J. Colloid Interface Sci. 1998, 198, 34.
residues on the protein surface and the negative charges on the functional groups of the support. In the case of Mb/ZrO2-BP, the main contribution is made by the hydrophobic interactions between the nonpolar side chains of the amino acid residues and the phenyl group on the surface of support. The kcat values decreased slightly as the amount of adsorbed protein onto ZrO2-P increased. This lowering of protein activity could have been caused by a possible diffusion barrier created by a dense packing of the myoglobin onto the surface of the support. The local protein concentration was higher than in free solution and protein-protein interactions limited the control of the orientation and packing of the biomolecules onto the surface. This resulted in the formation of a nonordered monolayer, which reduced the accessibility of the substrate to the active center as the amount of adsorbed protein increased. For the hydrophobic support ZrO2-BP, no difference was observed in kcat values as the amount of protein adsorbed increased. This highlights a difference orientation of the molecules onto the surfaces when hydrophobic interactions are involved. The KM value observed for the protein supported on nanoparticles was lower than that of free Mb indicating that there was a significant increase in the affinity of the protein for the substrate. It is interesting to note that the KM value increased, in comparison to that of free Mb, when Mb was immobilized on large-size R-ZrBP7 while decreased if Mb was immobilized on nanostructured ZrO2-BP. To explain these effects, it is suggested that the variation in affinity of substrate for the enzyme observed for the “large size” R-ZrBP compared to ZrO2-BP nanoparticles does not seem to be correlated to the hydrophobic nature of the support but to the great difference in sizes. A possible explanation could be related to the mobility of nanoparticles; the attached enzymes are not really “immobilized” but are similar to the native form. Further investigations are needed to understand the effect of support dimensions on the catalytic activity of immobilized enzyme. No significant differences were observed in the KM values between supports ZrO2-P and ZrO2-BP, indicating that the nature of the surface does not influence the affinity between the substrate and the enzyme. The variation in kcat and KM after immobilization can alter the catalytic efficiency, expressed by the kcat/KM ratios, in a different manner. The kcat/KM ratios of nanometric biocomposites were higher than those observed when Mb was immobilized onto large particles. This effect is mostly due to the value of KM that is lower in the nanobiocomposites. The kcat/KM ratios of nanoparticles were similar to that of free protein, and this was due to the decrease of kcat and KM for the nanobiocatalyst when compared to free protein. This result is of great interest because the higher affinity for the substrate allows the biocatalyst to react close to its maximum efficiency even at low substrate concentrations. The possibility of protein leaching from the support was tested in water and phosphate buffer (0.2 M, pH 6.0). The biocomposites were stirred in the aqueous medium for 5 min and then the mixture was centrifuged. The supernatant was assayed; no significant traces of activity were observed in any of the biocomposites obtained, showing that no appreciable desorption of protein from supports had occurred. The same experiment was carried out with the aromatic substrate used to evaluate the protein activity. The biocomposites were stirred in the presence
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Table 3. Peroxidase Activity of Mb Bound to ZrO2-P in the Oxidation of 2-Methoxyphenol by Hydrogen Peroxide (0.2 M Phosphate Buffer, pH 6.0, 25 °C) biocatalyst
Ca (µmol Mb/g supp)
[Mb]b (µM)
[H2O2]b (mM)
kcat (s-1)
KM (mM)
kcat/KM (mM-1 s-1)
free Mbc Mb/ZrO2-P(1) Mb/ZrO2-P(2)
4.8 11.6
1.0 1.2 1.4
67 342 570
5.5 ( 0.2 1.1 ( 0.1 (2.8 ( 0.1) 10-1
14.2 ( 1.6 5.2 ( 1.5 1.8 ( 0.6
(3.9 ( 0.5) 10-1 (2.1 ( 0.6) 10-1 (1.5 ( 0.4) 10-1
a
Amount of immobilized protein per unit mass of support. b Concentration inside of cuvette. c Reference 7.
Table 4. Peroxidase Activity of Mb Bound to ZrO2-BP in the Oxidation of 2-Methoxyphenol by Hydrogen Peroxide (0.2 M Phosphate Buffer, pH 6.0, 25 °C) biocatalyst
Ca (µmol Mb/g supp)
[Mb]b (µM)
[H2O2]b (mM)
kcat (s-1)
KM (mM)
kcat/KM (mM-1 s-1)
free Mbc Mb/R-ZrBPc Mb/ZrO2-BP(1) Mb/ZrO2-BP(2)
1.25 7.0 8.7
1.0 4.0 1.3 1.6
67 500 228 228
5.5 ( 0.2 (2.2 ( 0.3) 10-1 (2.3 ( 0.1) 10-1 (2.5 ( 0.1) 10-1
14.2 ( 1.6 35.8 ( 8.6 3.3 ( 0.7 2.4 ( 0.5
(3.9 ( 0.5) 10-1 (6.1 ( 1.6) 10-3 (7.1 ( 2.1) 10-2 (1.1 ( 0.2) 10-1
a
Amount of immobilized protein per unit mass of support. b Concentration inside of cuvette. c Reference 7.
of 2-methoxyphenol (30 mM in phosphate buffer 0.2 M, pH 6.0) for 5 min and then the mixture was centrifuged. The supernatant was assayed for activity by adding hydrogen peroxide and following the formation of oxidation product with UV-vis spectroscopy. No traces of activity were observed. Conclusions The nanometric colloidal particles obtained by grafting phosphate and phosphonate onto zirconia are good supports for the immobilization of myoglobin. The nanostructured biocomposites are active in the oxidation of 2-methoxyphenol by hydrogen peroxide, and the catalytic efficiency is similar to that of free Mb and higher than that of large-size biocatalysts. In the latter case, the values of kinetic parameters, kcat and KM, indicate that this effect is mostly due to an increased affinity of the nanobiocomposite for the substrate.
The activity of the nanobiocomposites decreased slightly as the amount of adsorbed protein increased. This was mainly due to the formation of a nonordered monolayer, which reduced the accessibility of the substrate to the active center. Furthermore, the activity of Mb immobilized onto nanoparticles could be related to their high mobility. All of these observations allow us to foresee that functionalized nanoparticles are optimal supports to immobilize proteins and enzymes. Acknowledgment. The authors express their gratitude to Prof. U. Costantino for helpful discussions and to Dott. F. Marmottini for measurement of the surface areas. LA051487Y
