- Email: [email protected]
Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles
International Journal of Biological Macromolecules 41 (2007) 404–409
Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles Rubens Araujo Silva, Ana M. Carmona-Ribeiro, Denise Freitas Siqueira Petri ∗ Instituto de Qu´ımica, Universidade de S˜ao Paulo, P.O. Box 26077, S˜ao Paulo, SP 05513-970, Brazil Received 2 April 2007; received in revised form 16 May 2007; accepted 24 May 2007 Available online 17 June 2007
Abstract The adsorption behavior of horseradish peroxidase (HRP) onto hybrid particles of poly(methylmethacrylate) (PMMA) and carboxymethylcellulose (CMC) was investigated by means of spectrophotometry. Dispersions of PMMA/CMC particles were characterized by light scattering, zeta potential measurements and scanning electron microscopy before and after HRP adsorption. HRP adsorbed irreversibly onto PMMA/CMC particles; the adsorption isotherm showed an initial step and an adsorption plateau. The enzymatic activity of free HRP and immobilized HRP (plateau region) was monitored by means of spectrophotometry as a function of storing time. Upon adsorbing HRP there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. HRP-covered PMMA/CMC particles dispersions, which were dried and re-dispersed, retained 50% of their catalytic properties. These interesting findings were discussed in the light of a beneficial effect of a hydrated microenvironment for maintenance of enzyme conformation and activity. © 2007 Elsevier B.V. All rights reserved. Keywords: Horseradish peroxidase; Carboxymethyl cellulose; Poly(methyl methacrylate); Adsorption; Activity
1. Introduction Enzyme immobilization onto solid surfaces brings the advantages of exploring catalytic properties in the desired reaction medium and, in some cases, of increasing enzyme stability [1]. On the other hand, the solid surface should be inert; it should not induce conformational changes upon enzyme adsorption. Peroxidase activity occurs in a large family of enzymes containing the heme (iron protoporphyrin) prosthetic group, which is responsible for catalysis in chemical reactions of the form: ROOR + electron donor (2e− ) + 2H+ → ROH + R OH For many of these enzymes the optimal substrate is hydrogen peroxide. Horseradish peroxidase (HRP) is a glycoprotein containing 21% carbohydrate with heme as prosthetic group, which requires hydrogen peroxide to achieve oxidation states [2,3]. Radical intermediates produced by HRP can oxidize a wide vari∗
Corresponding author. Tel.: +55 11 3091 3831; fax: +55 11 3818 5579. E-mail address: [email protected] (D.F.S. Petri).
0141-8130/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2007.05.014
ety of organic compounds [2,3]. Recently HRP has been used as catalyst for free radical polymerization and proved to be an interesting alternative for the conventional catalysts because the polymerization can be carried out at room temperature, yields reach acceptable levels and the solvent is not restricted to one special class, although organic solvents induce HRP activity loss [4]. Free HRP molecules catalyzed the polymerization of polyphenols [4], highly syndiotatic poly(methyl methacrylate) [5], polyacrylamide [6], polyaniline [7], polystyrene [8]. Not only free HRP molecules, but also immobilized HRP molecules can act as catalysts for free radical polymerization. Recently [9] HRP molecules immobilized onto Si wafers have been successfully used in the emulsion polymerization of poly(ethylglycol dimethacrylate) three times consecutively. The preservation of enzymatic activity of HRP adsorbed onto Si wafers was associated to the interface hydration, which avoids HRP denaturation after usage or upon storing. Other hydrophilic substrates proved to preserve HRP secondary structure after immobilization [10,11]. The activity and stability of HRP immobilized onto mesoporous silica with variable pore size were investigated [10]. The highest enzymatic activity and best thermal stability
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
were observed for HRP adsorbed onto silica with mean pore diameter of 5.0 nm. This pore size matches the HRP molecular dimensions, preventing enzymatic conformational changes, and therefore, keeping high activity and stability [10]. Monolayers of HRP adsorbed either onto Si wafers or onto succinylated modified Si wafers presented half-life time longer than 40 days at 6 ◦ C [11]. Moreover, HRP immobilization onto hydrophilic conductor materials has been applied for the development of amperometric sensors designed for hydrogen peroxide detection [12–14]. This work presents the adsorption behavior of HRP onto hybrid particles of poly(methyl methacrylate) (PMMA) and carboxymethyl cellulose (CMC) by means of spectrophotometry. PMMA/CMC hybrid particles are formed by PMMA core and CMC as outermost layers [15–17], providing hydrophilic carbohydrate rich surfaces. Such particles are excellent substrates for the adsorption of metallic ions [18] and lectins [19,20]. Particle characterization has been performed before and after HRP adsorption by means of dynamic light scattering, zeta potential measurements, scanning electron microscopy. The enzymatic activity of immobilized HRP was monitored by means of spectrophotometry, using a standard oxidation reaction, as a function of storing time. 2. Experimental 2.1. Materials Methyl methacrylate (MMA, Sigma, USA), cetyltrimethylammonium bromide (CTAB, Aldrich, USA), carboxymethyl cellulose (Sigma, USA, Mv ∼90,000 g mol−1 , DS = 0.7, Sigma, USA), potassium persulfate (CAAL, Brazil), horseradish peroxidase, HRP, type VI-A, from Amoracia rusticana (Sigma, USA, M ∼ 44,000 g mol−1 , P-6782, EC 1.11.1.7, 1380 units mg−1 ) and 2,2 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS, Sigma, USA) were used without further purification. HRP purity, also called Reinheitzahl (Rz), was estimated from the ratio of absorbance at 403 nm and 280 nm (Rz = A403 /A280 ) [21]. The Rz of HRP solution at 0.25 g L−1 amounted to 2.9 ± 0.1, indicating low contamination by other proteins [21]. 2.2. Synthesis of hybrid PMMA/CMC particles The synthesis of poly(methyl methacrylate), PMMA, in the presence of carboxymethyl cellulose, CMC, a cellulose derivative, was carried out by emulsion polymerization using a cationic surfactant, cetyltrimethylammonium bromide (CTAB). First, the complex formation between CTAB and CMC was studied by surface tension measurements [22]. The polymerization condition chosen was that corresponding to CMC chains fully saturated with CTAB and to the onset of pure surfactant micelles formation, namely at 0.25 mmol L−1 of CTAB and 1.0 g L−1 of CMC. The medium was purged with N2 during 30 min, while the temperature was brought to (82 ± 2) ◦ C. Afterwards the initiator, K2 S2 O8 , at the concentration of 3.3 g L−1 was added. Five minutes later MMA at the concentration of 66 g L−1 was
405
added to the system without any particular procedure. The polymerization was carried out under reflux and mechanical stirring (500 rpm). After 3 h the system was cooled to room temperature and dialysed (dialysis membrane 14,000 MW, Viskase Corporation, USA) against water with four changes daily during 1 week, or until the conductivity of dialysis water reached 5 S cm−1 . In this process no buffer was used. The dialysed dispersions presented pH in the range of 4.5–4.8. 2.3. Particle characterization The hybrid particles characterization was performed by means of a ZetaPlus-Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY) equipped with a 677 nm laser. The zeta potential value, ζ, was determined from the electrophoretic mobility, μ, in KCl 0.001 mol L−1 and Smoluchowski’s equation: ζ = μη/ε, where η is the medium viscosity and ε is the medium dielectric constant. The particle diameter D was obtained by dynamic light scattering at 90.0◦ . The dispersions were prepared in 0.001 mol L−1 NaCl. The particle size distribution from analysis of quasielastic light scattering (QELS) data was performed following well-established mathematical techniques [23]. Field emission scanning electron microscopy (SEM-FEG) was performed in a JEOL JSM-7000F microscope on droplets of dispersions at 2.8 × 1011 particles mL−1 after drying in air. The solid content and the conversion of monomer into polymer were determined by gravimetry. The mean particle number density (Np ) was calculated considering the particle mean diameter determined by QELS, solid content in 1.00 mL of dispersion and polymer density as 1.00 g cm−3 . 2.4. Adsorption of HRP onto PMMA/CMC particles HRP solutions in the concentration range of 12.5 mg mL−1 to 300 mg mL−1 in NaCl 1.0 mmol L−1 were added to dispersions of PMMA/CMC previously diluted in NaCl 1.0 mmol L−1 at the concentration of 2.8 × 1011 particles mL−1 at (24 ± 1) ◦ C. After 4 h the mixture was centrifuged at 14,000 rpm for 30 min. The particles were carefully separated from the supernatant. The concentration of free HRP in the supernatant was determined by UV-spectrophotometry at 403 nm in a Beckmann-Coulter DU600 spectrophotometer, for the optical pathway length as 1 cm and HRP extinction coefficient ε of 1.57 g−1 L cm−1 (or 35.7 mol−1 L cm−1 ), as determined from a calibration curve (supplementary material). The difference between this value and the initial HRP concentration yielded the concentration of adsorbed HRP, [HRP]ads . All experiments were carried out at (24 ± 1) ◦ C. Dispersions of HRP-covered PMMA/CMC particles were characterized by light scattering, zeta potential measurements and SEM-FEG. Desorption experiments were carried out by re-dispersing HRP-covered PMMA/CMC particles in NaCl 1.0 mmol L−1 , so that Np was 2.8 × 1011 particles mL−1 . After 24 h the dispersions were centrifuged at 14,000 rpm for 30 min and the concentration of free HRP in the supernatant was determined by UV-spectrophotometry at 403 nm.
406
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
2.5. Catalytic activity of adsorbed HRP and free HRP The catalytic activity of adsorbed HRP and free HRP was tested based on the oxidation of ABTS catalyzed by HRP in the presence of H2 O2 , into the radical cation ABTS•+ [24,25], a standard reaction to evaluate HRP enzymatic activity. The concentration of free HRP was the same as [HRP]ads . The oxidation of ABTS into ABTS•+ (λmax = 414 nm) was monitored by UV–vis spectrophotometry (Beckmann-Coulter DU 600) as a function of time. The concentration of ABTS•+ was calculated using Beer–Lambert equation for the optical pathway of 1 cm and extinction coefficient of 3.6 × 104 mol L−1 cm−1 [24]. The formation of ABTS•+ was evidenced by the appearance of green color solution. Control experiments were done using free HRP in solution under the same conditions and reactants amounts. In another set of experiments dispersions of HRP-covered PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) were dried at 24 ± 1 ◦ C during 72 h or until constant weight. After that the dried HRP-covered PMMA/CMC particles were redispersed in NaCl 1.0 mmol L−1 to the original volume at 24 ± 1 ◦ C. After 48 h catalytic activity of re-dispersed HRPcovered PMMA/CMC particles was measured with basis on ABTS oxidation. 3. Results and discussion 3.1. Adsorption of HRP onto PMMA/CMC particles PMMA/CMC particles presented mean zeta potential value, ζ, of −(46 ± 3) mV, indicating that the CMC chains are on the particle surface with the carboxylate groups oriented to the aqueous medium [15,16]. In Fig. 1a particle size distribution determined in NaCl 0.001 mol L−1 by means of DLS presented two peaks, which correspond to a large population with particle mean diameter of (145 ± 10) nm and a small population with particle mean diameter of (295 ± 8) nm. The second peak might be attributed to doublets of particles. However, in order to estimate the mean particle number density (Np ) the particle mean diameter of (145 ± 10) nm has been chosen because it corresponded to the most populated peak. The estimate of Np also considered polymer density of 1.00 g cm−3 and solid content of (9 ± 1) g L−1 . Np was calculated of 5.6 × 1012 particles mL−1 . SEM-FEG images (Fig. 1b) of dried PMMA/CMC particles indicated mean particle diameter of (110 ± 10) nm, which is less than the mean particle size determined by means of DLS. Such difference stems from drying process, which leads to particles collapse. Recently [19] the thickness of CMC outermost layer of PMMA/CMC particles in water was determined as (20 ± 10) nm by means of adhesion forces. Upon drying the CMC outermost layer collapses, reducing the mean particle diameter. The adsorption isotherm of HRP onto PMMA/CMC hybrid particles presented in Fig. 2 can be divided into two regions: (i) an initial increase of the adsorbed amount with HRP concentration and (ii) an adsorption plateau at [HRP]ads = (0.022 ± 0.002) g L−1 with onset of 0.10 g L−1 of [HRP]ini . The initial step is commonly observed in the very dilute range of adsorbate. The adsorbed amount
Fig. 1. (a) Particle size distribution obtained for dispersions of PMMA/CMC prepared at 2.8 × 1011 particles mL−1 . (b) Scanning electron microscopy obtained for dried PMMA/CMC particles.
Fig. 2. Adsorption isotherm obtained for HRP onto PMMA/CMC particles at 2.8 × 1011 particles mL−1 and (24 ± 1) ◦ C.
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
increases up to the substrate saturation. The adsorption plateau might correspond to the formation of a monolayer. Taking the [HRP]ads = (0.022 ± 0.002) g L−1 , HRP molecular weight of 44,000 g mol−1 , the Avogadro number (6.02 × 1023 molecules mol−1 ), and Np 2.8 × 1014 particles L−1 , one can estimate ∼1000 HRP molecules adsorbed onto one PMMA/CMC particle. The mean surface area (4πr2 ) of one PMMA/CMC particle amounts to ∼66,000 nm2 . The radius of gyration of HRP monomer was calculated by dynamics simulations [26] as 2.65 nm, assuming the crystallographic dimensions of 4.0 nm × 6.7 nm × 11.7 nm [27]. Considering HRP as a solid sphere, the surface area of one HRP amounts to ∼88 nm2 . Therefore, the area of 1000 HRP molecules amounts to 88,000 nm2 , which is in the same order of magnitude of the area of one PMMA/CMC particle. These estimates are not accurate because the real area of one PMMA/CMC particle is probably larger than the calculated one, since CMC loops and trains on the particle surface were not considered, and HRP is not a perfect sphere. However, they evidence the monolayer formation at the adsorption plateau. HRP-covered PMMA/CMC particles presented mean diameter and zeta potential values of (143 ± 2) nm and −(45 ± 3) mV, respectively, such findings are expected since HRP molecules are much smaller than PMMA/CMC particles and HRP molecules are composed of charged and uncharged residues. According to PDB data HRP is a glycoprotein containing 21% carbohydrate with heme as prosthetic group [2,3] and 308 amino acid residues (60% of hydrophilic residues and 40% of hydrophobic residues). Desorption experiments showed that HRP molecules adsorb irreversibly onto PMMA/CMC hybrid particles. All adsorption experiments were performed at pH 6.5, which is the optimum pH of HRP [28]. Under such condition PMMA/CMC particles are negatively charged, as evidenced by zeta potential values, due to the carboxylate groups exposed to the medium. HRP net charge under pH 6.5 is unknown because HRP presents at least seven isozymes, with isoelectric point ranging from 3.0 to 9.0 [29]. However, favorable interactions between carbohydrate segments of CMC outermost layers and HRP carbohydrate and/or amino acid residues are expected, which might cause irreversible adsorption.
407
Fig. 3. Photograph of dispersions of (A) HRP-covered PMMA/CMA hybrid particles (Np 2.8 × 1011 particles mL−1 and [HRP]ads = 0.022 g L−1 ) and (B) bare PMMA/CMC hybrid particles (Np 2.8 × 1011 particles mL−1 ) under reaction medium composed of H2 O2 and ABTS.
ABTS•+ is much faster when HRP molecules are free in solution than when HRP molecules are immobilized. The initial steps (up to 50 s) could be fitted with linear regression y = 0.418 (R = 0.9297) and y = 0.206 (R = 0.9879), when the ABTS•+ formation was catalyzed by free HRP and immobilized HRP, respectively. The access for the substrate is more difficult when HRP molecules are immobilized than when they are free in solution. In other words the rate-limiting step of the reaction is substrate diffusion within the heterogeneous catalyst (immobilized HRP). After 10 min of reaction one notices that when HRP molecules are free in solution the oxidation amount of product formed (21.0 ± 0.3 mmol L−1 of ABTS•+ ) is only 10% higher than that observed when HRP molecules are immobilized (18.5 ± 0.5 mmol L−1 of ABTS•+ ). How storing time affects the enzymatic activity is a persistent question in many industrial processes. Freshly prepared solu-
3.2. Catalytic activity of adsorbed HRP and free HRP The enzymatic activity of immobilized HRP onto PMMA/ CMC was investigated in the presence of H2 O2 , by means of oxidation of ABTS into the radical cation ABTS•+ [23,24] and compared to that of free HRP under the same conditions. The experimental condition chosen was that corresponding to the adsorption plateau, [HRP]ads = (0.022 ± 0.002) g L−1 . The green color characteristic of this reaction was observed visually (Fig. 3A) for HRP-covered PMMA/CMC particles, indicating that immobilized HRP molecules retained their catalytic properties. As a control, the green color was absent, indicating no oxidation of ABTS in the absence of HRP (Fig. 3B). Fig. 4 shows the formation of radical cation ABTS•+ in the presence of H2 O2 catalyzed by free HRP or immobilized HRP at 0.022 g L−1 , as a function of time. The formation of
Fig. 4. Formation of radical cation ABTS•+ (mol L−1 ) in the presence of H2 O2 catalyzed by free HRP at 0.022 g L−1 (䊉) or immobilized HRP ([HRP]ads = 0.022 g L−1 ) onto PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) ()), as a function of time.
408
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
Fig. 5. Enzymatic activity of free HRP at 0.022 g L−1 (white column) and immobilized HRP ([HRP]ads = 0.022 g L−1 ) onto PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) (patterned column), expressed in terms of concentration of radical cation ABTS•+ formed after 10 min of reaction, as a function of storing time.
tions of free HRP and HRP-covered PMMA/CMC particles were stored in closed vials at (24 ± 1) ◦ C for different periods of time and after each period of storing time the catalytic activity of free and immobilized HRP was determined. For the sake of comparison the concentration of free and immobilized HRP was the same and corresponded to that at the adsorption plateau, namely, [HRP]ads = 0.022 g L−1 . Fig. 5 shows the enzymatic activity of free HRP and immobilized HRP, expressed in terms of concentration of radical cation ABTS•+ formed after 10 min of reaction, as a function of storing time. One observes that upon adsorbing HRP, there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. These findings show that (i) storing did not cause changes in the HRP secondary structure and (ii) the surface of PMMA/CMC hybrid particles provided an environment, which prevented HRP conformational changes. Recently [19], measurements of adhesion forces have shown that PMMA/CMC hybrid particles present a hard core and a fluffy layer (20 ± 10) nm thick, which was attributed to the highly hydrated CMC outermost layer around the particle. Similarly HRP molecules presented no reduction of enzymatic activity when they were entrapped in mesoporous silica particles [10], which are very hydrophilic surfaces [30]. Similar behavior for other enzymes can be found in the literature. The catalytic behavior of creatine phosphokinase (CPK) immobilized onto Si/SiO2 wafers under different pH conditions showed that under alkaline conditions, the contribution of strong hydration forces weakened CPK unfolding upon electrostatically driven adsorption [31]. The preservation of enzymatic activity of hexokinase adsorbed onto Si/SiO2 wafers was associated to the interface hydration, which avoided hexokinase denaturation after usage or upon storing [32]. High hydration level on the surface seems to play a very important role, preserving enzyme conformation upon adsorption. Colloidal particles of block copolymer poly(styreneb-acrylic acid), PS-b-PAA, were used for the immobilization of pectinase [33]. The carboxyl groups on the particles surface
Fig. 6. Enzymatic activity of immobilized HRP ([HRP]ads = 0.025 g L−1 ) onto PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) freshly prepared () and after drying and re-dispersion () expressed in terms of concentration of radical cation ABTS•+ formed after 10 min of reaction, as a function of time.
led to improved thermal and storage stabilities of immobilized pectinase. Microspheres of PMMA functionalized with acrolein were used for the immobilization of trypsin with successful performance, since increase in thermal stability, pH stability and storage lifetime has been observed [34]. Another interesting result was observed when dispersions of HRP-covered PMMA/CMC particles were dried at 24 ± 1 ◦ C during 72 h and re-dispersed in NaCl 0.001 mol L−1 at the same temperature. The re-dispersed HRP-covered PMMA/CMC particles retained 50% of their original enzymatic activity (Fig. 6), showing that drying brought about conformational changes in HRP structure. Many of water molecules around CMC layers are removed upon drying, perturbing HRP native structure. This finding evidences the crucial role played by surface hydration on the activity of immobilized enzymes. There are interesting reports in the literature [35–37], which show that hydrophobic polystyrene particles are not appropriate for enzyme immobilization. Recently Koutsopoulos et al. [35] observed that upon adsorbing trypsin onto PS films the enzymatic activity was completely suppressed. Caruso et al. [36,37] modified PS latex particles using layer-by-layer polyelectrolyte adsorption technique to facilitate subsequent enzyme adsorption. Four alternating poly(allylamine hydrochloride), PAH, and poly(styrenesulfonate), PSS, layers were deposited onto the PS lattices (first layer PAH), resulting in negatively charged particles with PSS as the outermost layer. The enzyme beta glucosidase [36] or glucose oxidase [37] was then adsorbed onto the outermost PSS layer. The enzyme multilayer-coated particles were enzymatically active, with the general finding that the total activity of the particles increased with increasing enzyme layer number. 4. Conclusions HRP adsorbed irreversibly onto PMMA/CMC hybrid particles, probably due to favorable interaction between HRP carbohydrate residues and CMC segments. HRP-covered PMMA/CMC particles work as catalytic devices with performance comparable to that of free HRP molecules. Such particles can be stored for long terms under room temperature
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
without enzymatic activity loss. Moreover, dried HRP-covered PMMA/CMC particles can be added to the desired reaction yielding 50% of original enzymatic activity. Preservation of enzyme activity on PMMA/CMC was attributed to the presence of hydrated CMC layers around the particles. Acknowledgments The authors acknowledge FAPESP and CNPq for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijbiomac.2007.05.014. References [1] I. Chibata, T. Tosa, T. Sato, T. Mori, Immobilized Enzymes, Wiley & Sons, New York, 1978. [2] N.C. Veitch, Phytochemistry 65 (2004) 249–259. [3] G.I. Berglund, G.H. Carlsson, A.T. Smith, H. Sz¨oke, A. Henriksen, J. Hajdu, Nature 417 (2002) 463–468. [4] J.S. Dordick, M.A. Marletta, A.M. Klibanov, Biotechnol. Bioeng. 30 (1987) 31–36. [5] B. Kalra, R.A. Gross, Biomacromolecules 1 (2000) 501–505. [6] B. Kalra, R.A. Gross, Green Chem. 4 (2002) 174–178. [7] W. Liu, J. Kumar, S. Tripathy, K.J. Senecal, L. Samuelson, J. Am. Chem. Soc. 121 (1999) 71–78. [8] J. Shan, Y. Kitamura, H. Yoshizawa, Colloid Polym. Sci. 284 (2005) 108–111. [9] A.F. Naves, A.M. Carmona-Ribeiro, D.F.S. Petri, Langmuir 23 (2007) 1981–1987. [10] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Chem. Mater. 12 (2000) 3301–3305. [11] F. Vianello, L. Zennaro, M.L. Di Paolo, A. Rigo, C. Malacarne, M. Scarpa, Biotechnol. Bioeng. 68 (2000) 488–495. [12] S. Yabuki, F. Mizutami, Y. Hirata, Sens. Actuators B: Chem. 65 (2000) 46–48.
409
[13] G.H. Wang, L.M. Zhang, J. Phys. Chem. B 110 (2006) 24864–24868. [14] N.Q. Jia, J. Xu, H.M. Sun, Z.Y. Jiang, Anal. Lett. 38 (2005) 123–124. [15] L.B.R. Castro, K.V. Soares, A.F. Naves, A.M. Carmona-Ribeiro, D.F.S. Petri, Ind. Eng. Chem. Res. 43 (2004) 7774–7779. [16] A.F. Naves, P.M. Kosaka, J.R. Matos, D.F.S. Petri, J. Therm. Anal. Calorim. 79 (2005) 389–392. [17] A.M. Carmona-Ribeiro, D.F.S. Petri, in: H.S. Nalwa (Ed.), Polymeric Nanostructures and their Applications, vol. 1, American Scientific Publishers, California, 2007, pp. 485–529. [18] K.V. Soares, J.C. Masini, R.M. Torresi, A.M. Carmona-Ribeiro, D.F.S. Petri, Langmuir 21 (2005) 8515–8519. [19] L.B.R. Castro, M. Kappl, D.F.S. Petri, Langmuir 22 (2006) 3757– 3762. [20] L.B.R. Castro, D.F.S. Petri, J. Nanosci. Nanotech. 5 (2005) 2063–2069. [21] K. Chattopadhyay, S. Mazumdar, Biochemistry 39 (2000) 263–270. [22] A.F. Naves, D.F.S. Petri, Colloid Surf. A: Physicochem. Eng. Aspects 254 (2005) 207–214. [23] E.F. Grabowski, I.D. Morrison, in: B. Dahneke (Ed.), Measurements of Suspended Particles by Quasi-Elastic Light Scattering, Wiley–Interscience, New York, 1983 (chapter 7). [24] E.N. Kadnikova, N.M. Kostic, J. Mol. Catal. B: Enzym. 18 (2002) 39–48. [25] R.E. Childs, W.G. Bardsley, Biochem. J. 145 (1975) 93–103. [26] M. Laberge, Q. Huang, R. Schweitzer-Stenner, J. Fidy, Biophys. J. 84 (2003) 2542–2552. [27] http://www.rcsb.org/pdb ID: 1H58, accessed in January 17, 2007. [28] D. Schomberg, M. Salzmann, D. Stephan, Enzyme Handbook, vol. 7, 1993, EC 1.11.1.7:1–6. [29] L.M. Shannon, E. Kay, J.Y. Lew, J. Biol. Chem. 241 (1966) 2166–2168. [30] L.T. Zhuravlev, Colloid Surf. A: Physicochem. Eng. Aspects 173 (2000) 1–38. [31] S.M. Pancera, H. Gliemann, T. Schimmel, D.F.S. Petri, J. Phys. Chem. B 110 (2006) 2674–2680. [32] S.M. Pancera, H. Gliemann, T. Schimmel, D.F.S. Petri, J. Colloid Interf. Sci. 302 (2006) 417–423. [33] Z. Lei, S. Bi, J. Biotechnol. 128 (2007) 112–119. [34] S. Wu, B. Liu, S. Li, Int. J. Biol. Macromol. 37 (2005) 263–267. [35] S. Koutsopoulos, K. Patzsch, W.T.E. Bosker, W. Norde, Langmuir 23 (2007) 2000–2006. [36] F. Caruso, H. Fiedler, K. Haage, Colloid Surf. A: Physicochem. Eng. Aspects 169 (2000) 287–293. [37] F. Caruso, C. Sch¨uler, Langmuir 16 (2000) 9595–9603.
Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles Rubens Araujo Silva, Ana M. Carmona-Ribeiro, Denise Freitas Siqueira Petri ∗ Instituto de Qu´ımica, Universidade de S˜ao Paulo, P.O. Box 26077, S˜ao Paulo, SP 05513-970, Brazil Received 2 April 2007; received in revised form 16 May 2007; accepted 24 May 2007 Available online 17 June 2007
Abstract The adsorption behavior of horseradish peroxidase (HRP) onto hybrid particles of poly(methylmethacrylate) (PMMA) and carboxymethylcellulose (CMC) was investigated by means of spectrophotometry. Dispersions of PMMA/CMC particles were characterized by light scattering, zeta potential measurements and scanning electron microscopy before and after HRP adsorption. HRP adsorbed irreversibly onto PMMA/CMC particles; the adsorption isotherm showed an initial step and an adsorption plateau. The enzymatic activity of free HRP and immobilized HRP (plateau region) was monitored by means of spectrophotometry as a function of storing time. Upon adsorbing HRP there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. HRP-covered PMMA/CMC particles dispersions, which were dried and re-dispersed, retained 50% of their catalytic properties. These interesting findings were discussed in the light of a beneficial effect of a hydrated microenvironment for maintenance of enzyme conformation and activity. © 2007 Elsevier B.V. All rights reserved. Keywords: Horseradish peroxidase; Carboxymethyl cellulose; Poly(methyl methacrylate); Adsorption; Activity
1. Introduction Enzyme immobilization onto solid surfaces brings the advantages of exploring catalytic properties in the desired reaction medium and, in some cases, of increasing enzyme stability [1]. On the other hand, the solid surface should be inert; it should not induce conformational changes upon enzyme adsorption. Peroxidase activity occurs in a large family of enzymes containing the heme (iron protoporphyrin) prosthetic group, which is responsible for catalysis in chemical reactions of the form: ROOR + electron donor (2e− ) + 2H+ → ROH + R OH For many of these enzymes the optimal substrate is hydrogen peroxide. Horseradish peroxidase (HRP) is a glycoprotein containing 21% carbohydrate with heme as prosthetic group, which requires hydrogen peroxide to achieve oxidation states [2,3]. Radical intermediates produced by HRP can oxidize a wide vari∗
Corresponding author. Tel.: +55 11 3091 3831; fax: +55 11 3818 5579. E-mail address: [email protected] (D.F.S. Petri).
0141-8130/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2007.05.014
ety of organic compounds [2,3]. Recently HRP has been used as catalyst for free radical polymerization and proved to be an interesting alternative for the conventional catalysts because the polymerization can be carried out at room temperature, yields reach acceptable levels and the solvent is not restricted to one special class, although organic solvents induce HRP activity loss [4]. Free HRP molecules catalyzed the polymerization of polyphenols [4], highly syndiotatic poly(methyl methacrylate) [5], polyacrylamide [6], polyaniline [7], polystyrene [8]. Not only free HRP molecules, but also immobilized HRP molecules can act as catalysts for free radical polymerization. Recently [9] HRP molecules immobilized onto Si wafers have been successfully used in the emulsion polymerization of poly(ethylglycol dimethacrylate) three times consecutively. The preservation of enzymatic activity of HRP adsorbed onto Si wafers was associated to the interface hydration, which avoids HRP denaturation after usage or upon storing. Other hydrophilic substrates proved to preserve HRP secondary structure after immobilization [10,11]. The activity and stability of HRP immobilized onto mesoporous silica with variable pore size were investigated [10]. The highest enzymatic activity and best thermal stability
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
were observed for HRP adsorbed onto silica with mean pore diameter of 5.0 nm. This pore size matches the HRP molecular dimensions, preventing enzymatic conformational changes, and therefore, keeping high activity and stability [10]. Monolayers of HRP adsorbed either onto Si wafers or onto succinylated modified Si wafers presented half-life time longer than 40 days at 6 ◦ C [11]. Moreover, HRP immobilization onto hydrophilic conductor materials has been applied for the development of amperometric sensors designed for hydrogen peroxide detection [12–14]. This work presents the adsorption behavior of HRP onto hybrid particles of poly(methyl methacrylate) (PMMA) and carboxymethyl cellulose (CMC) by means of spectrophotometry. PMMA/CMC hybrid particles are formed by PMMA core and CMC as outermost layers [15–17], providing hydrophilic carbohydrate rich surfaces. Such particles are excellent substrates for the adsorption of metallic ions [18] and lectins [19,20]. Particle characterization has been performed before and after HRP adsorption by means of dynamic light scattering, zeta potential measurements, scanning electron microscopy. The enzymatic activity of immobilized HRP was monitored by means of spectrophotometry, using a standard oxidation reaction, as a function of storing time. 2. Experimental 2.1. Materials Methyl methacrylate (MMA, Sigma, USA), cetyltrimethylammonium bromide (CTAB, Aldrich, USA), carboxymethyl cellulose (Sigma, USA, Mv ∼90,000 g mol−1 , DS = 0.7, Sigma, USA), potassium persulfate (CAAL, Brazil), horseradish peroxidase, HRP, type VI-A, from Amoracia rusticana (Sigma, USA, M ∼ 44,000 g mol−1 , P-6782, EC 1.11.1.7, 1380 units mg−1 ) and 2,2 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS, Sigma, USA) were used without further purification. HRP purity, also called Reinheitzahl (Rz), was estimated from the ratio of absorbance at 403 nm and 280 nm (Rz = A403 /A280 ) [21]. The Rz of HRP solution at 0.25 g L−1 amounted to 2.9 ± 0.1, indicating low contamination by other proteins [21]. 2.2. Synthesis of hybrid PMMA/CMC particles The synthesis of poly(methyl methacrylate), PMMA, in the presence of carboxymethyl cellulose, CMC, a cellulose derivative, was carried out by emulsion polymerization using a cationic surfactant, cetyltrimethylammonium bromide (CTAB). First, the complex formation between CTAB and CMC was studied by surface tension measurements [22]. The polymerization condition chosen was that corresponding to CMC chains fully saturated with CTAB and to the onset of pure surfactant micelles formation, namely at 0.25 mmol L−1 of CTAB and 1.0 g L−1 of CMC. The medium was purged with N2 during 30 min, while the temperature was brought to (82 ± 2) ◦ C. Afterwards the initiator, K2 S2 O8 , at the concentration of 3.3 g L−1 was added. Five minutes later MMA at the concentration of 66 g L−1 was
405
added to the system without any particular procedure. The polymerization was carried out under reflux and mechanical stirring (500 rpm). After 3 h the system was cooled to room temperature and dialysed (dialysis membrane 14,000 MW, Viskase Corporation, USA) against water with four changes daily during 1 week, or until the conductivity of dialysis water reached 5 S cm−1 . In this process no buffer was used. The dialysed dispersions presented pH in the range of 4.5–4.8. 2.3. Particle characterization The hybrid particles characterization was performed by means of a ZetaPlus-Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY) equipped with a 677 nm laser. The zeta potential value, ζ, was determined from the electrophoretic mobility, μ, in KCl 0.001 mol L−1 and Smoluchowski’s equation: ζ = μη/ε, where η is the medium viscosity and ε is the medium dielectric constant. The particle diameter D was obtained by dynamic light scattering at 90.0◦ . The dispersions were prepared in 0.001 mol L−1 NaCl. The particle size distribution from analysis of quasielastic light scattering (QELS) data was performed following well-established mathematical techniques [23]. Field emission scanning electron microscopy (SEM-FEG) was performed in a JEOL JSM-7000F microscope on droplets of dispersions at 2.8 × 1011 particles mL−1 after drying in air. The solid content and the conversion of monomer into polymer were determined by gravimetry. The mean particle number density (Np ) was calculated considering the particle mean diameter determined by QELS, solid content in 1.00 mL of dispersion and polymer density as 1.00 g cm−3 . 2.4. Adsorption of HRP onto PMMA/CMC particles HRP solutions in the concentration range of 12.5 mg mL−1 to 300 mg mL−1 in NaCl 1.0 mmol L−1 were added to dispersions of PMMA/CMC previously diluted in NaCl 1.0 mmol L−1 at the concentration of 2.8 × 1011 particles mL−1 at (24 ± 1) ◦ C. After 4 h the mixture was centrifuged at 14,000 rpm for 30 min. The particles were carefully separated from the supernatant. The concentration of free HRP in the supernatant was determined by UV-spectrophotometry at 403 nm in a Beckmann-Coulter DU600 spectrophotometer, for the optical pathway length as 1 cm and HRP extinction coefficient ε of 1.57 g−1 L cm−1 (or 35.7 mol−1 L cm−1 ), as determined from a calibration curve (supplementary material). The difference between this value and the initial HRP concentration yielded the concentration of adsorbed HRP, [HRP]ads . All experiments were carried out at (24 ± 1) ◦ C. Dispersions of HRP-covered PMMA/CMC particles were characterized by light scattering, zeta potential measurements and SEM-FEG. Desorption experiments were carried out by re-dispersing HRP-covered PMMA/CMC particles in NaCl 1.0 mmol L−1 , so that Np was 2.8 × 1011 particles mL−1 . After 24 h the dispersions were centrifuged at 14,000 rpm for 30 min and the concentration of free HRP in the supernatant was determined by UV-spectrophotometry at 403 nm.
406
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
2.5. Catalytic activity of adsorbed HRP and free HRP The catalytic activity of adsorbed HRP and free HRP was tested based on the oxidation of ABTS catalyzed by HRP in the presence of H2 O2 , into the radical cation ABTS•+ [24,25], a standard reaction to evaluate HRP enzymatic activity. The concentration of free HRP was the same as [HRP]ads . The oxidation of ABTS into ABTS•+ (λmax = 414 nm) was monitored by UV–vis spectrophotometry (Beckmann-Coulter DU 600) as a function of time. The concentration of ABTS•+ was calculated using Beer–Lambert equation for the optical pathway of 1 cm and extinction coefficient of 3.6 × 104 mol L−1 cm−1 [24]. The formation of ABTS•+ was evidenced by the appearance of green color solution. Control experiments were done using free HRP in solution under the same conditions and reactants amounts. In another set of experiments dispersions of HRP-covered PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) were dried at 24 ± 1 ◦ C during 72 h or until constant weight. After that the dried HRP-covered PMMA/CMC particles were redispersed in NaCl 1.0 mmol L−1 to the original volume at 24 ± 1 ◦ C. After 48 h catalytic activity of re-dispersed HRPcovered PMMA/CMC particles was measured with basis on ABTS oxidation. 3. Results and discussion 3.1. Adsorption of HRP onto PMMA/CMC particles PMMA/CMC particles presented mean zeta potential value, ζ, of −(46 ± 3) mV, indicating that the CMC chains are on the particle surface with the carboxylate groups oriented to the aqueous medium [15,16]. In Fig. 1a particle size distribution determined in NaCl 0.001 mol L−1 by means of DLS presented two peaks, which correspond to a large population with particle mean diameter of (145 ± 10) nm and a small population with particle mean diameter of (295 ± 8) nm. The second peak might be attributed to doublets of particles. However, in order to estimate the mean particle number density (Np ) the particle mean diameter of (145 ± 10) nm has been chosen because it corresponded to the most populated peak. The estimate of Np also considered polymer density of 1.00 g cm−3 and solid content of (9 ± 1) g L−1 . Np was calculated of 5.6 × 1012 particles mL−1 . SEM-FEG images (Fig. 1b) of dried PMMA/CMC particles indicated mean particle diameter of (110 ± 10) nm, which is less than the mean particle size determined by means of DLS. Such difference stems from drying process, which leads to particles collapse. Recently [19] the thickness of CMC outermost layer of PMMA/CMC particles in water was determined as (20 ± 10) nm by means of adhesion forces. Upon drying the CMC outermost layer collapses, reducing the mean particle diameter. The adsorption isotherm of HRP onto PMMA/CMC hybrid particles presented in Fig. 2 can be divided into two regions: (i) an initial increase of the adsorbed amount with HRP concentration and (ii) an adsorption plateau at [HRP]ads = (0.022 ± 0.002) g L−1 with onset of 0.10 g L−1 of [HRP]ini . The initial step is commonly observed in the very dilute range of adsorbate. The adsorbed amount
Fig. 1. (a) Particle size distribution obtained for dispersions of PMMA/CMC prepared at 2.8 × 1011 particles mL−1 . (b) Scanning electron microscopy obtained for dried PMMA/CMC particles.
Fig. 2. Adsorption isotherm obtained for HRP onto PMMA/CMC particles at 2.8 × 1011 particles mL−1 and (24 ± 1) ◦ C.
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
increases up to the substrate saturation. The adsorption plateau might correspond to the formation of a monolayer. Taking the [HRP]ads = (0.022 ± 0.002) g L−1 , HRP molecular weight of 44,000 g mol−1 , the Avogadro number (6.02 × 1023 molecules mol−1 ), and Np 2.8 × 1014 particles L−1 , one can estimate ∼1000 HRP molecules adsorbed onto one PMMA/CMC particle. The mean surface area (4πr2 ) of one PMMA/CMC particle amounts to ∼66,000 nm2 . The radius of gyration of HRP monomer was calculated by dynamics simulations [26] as 2.65 nm, assuming the crystallographic dimensions of 4.0 nm × 6.7 nm × 11.7 nm [27]. Considering HRP as a solid sphere, the surface area of one HRP amounts to ∼88 nm2 . Therefore, the area of 1000 HRP molecules amounts to 88,000 nm2 , which is in the same order of magnitude of the area of one PMMA/CMC particle. These estimates are not accurate because the real area of one PMMA/CMC particle is probably larger than the calculated one, since CMC loops and trains on the particle surface were not considered, and HRP is not a perfect sphere. However, they evidence the monolayer formation at the adsorption plateau. HRP-covered PMMA/CMC particles presented mean diameter and zeta potential values of (143 ± 2) nm and −(45 ± 3) mV, respectively, such findings are expected since HRP molecules are much smaller than PMMA/CMC particles and HRP molecules are composed of charged and uncharged residues. According to PDB data HRP is a glycoprotein containing 21% carbohydrate with heme as prosthetic group [2,3] and 308 amino acid residues (60% of hydrophilic residues and 40% of hydrophobic residues). Desorption experiments showed that HRP molecules adsorb irreversibly onto PMMA/CMC hybrid particles. All adsorption experiments were performed at pH 6.5, which is the optimum pH of HRP [28]. Under such condition PMMA/CMC particles are negatively charged, as evidenced by zeta potential values, due to the carboxylate groups exposed to the medium. HRP net charge under pH 6.5 is unknown because HRP presents at least seven isozymes, with isoelectric point ranging from 3.0 to 9.0 [29]. However, favorable interactions between carbohydrate segments of CMC outermost layers and HRP carbohydrate and/or amino acid residues are expected, which might cause irreversible adsorption.
407
Fig. 3. Photograph of dispersions of (A) HRP-covered PMMA/CMA hybrid particles (Np 2.8 × 1011 particles mL−1 and [HRP]ads = 0.022 g L−1 ) and (B) bare PMMA/CMC hybrid particles (Np 2.8 × 1011 particles mL−1 ) under reaction medium composed of H2 O2 and ABTS.
ABTS•+ is much faster when HRP molecules are free in solution than when HRP molecules are immobilized. The initial steps (up to 50 s) could be fitted with linear regression y = 0.418 (R = 0.9297) and y = 0.206 (R = 0.9879), when the ABTS•+ formation was catalyzed by free HRP and immobilized HRP, respectively. The access for the substrate is more difficult when HRP molecules are immobilized than when they are free in solution. In other words the rate-limiting step of the reaction is substrate diffusion within the heterogeneous catalyst (immobilized HRP). After 10 min of reaction one notices that when HRP molecules are free in solution the oxidation amount of product formed (21.0 ± 0.3 mmol L−1 of ABTS•+ ) is only 10% higher than that observed when HRP molecules are immobilized (18.5 ± 0.5 mmol L−1 of ABTS•+ ). How storing time affects the enzymatic activity is a persistent question in many industrial processes. Freshly prepared solu-
3.2. Catalytic activity of adsorbed HRP and free HRP The enzymatic activity of immobilized HRP onto PMMA/ CMC was investigated in the presence of H2 O2 , by means of oxidation of ABTS into the radical cation ABTS•+ [23,24] and compared to that of free HRP under the same conditions. The experimental condition chosen was that corresponding to the adsorption plateau, [HRP]ads = (0.022 ± 0.002) g L−1 . The green color characteristic of this reaction was observed visually (Fig. 3A) for HRP-covered PMMA/CMC particles, indicating that immobilized HRP molecules retained their catalytic properties. As a control, the green color was absent, indicating no oxidation of ABTS in the absence of HRP (Fig. 3B). Fig. 4 shows the formation of radical cation ABTS•+ in the presence of H2 O2 catalyzed by free HRP or immobilized HRP at 0.022 g L−1 , as a function of time. The formation of
Fig. 4. Formation of radical cation ABTS•+ (mol L−1 ) in the presence of H2 O2 catalyzed by free HRP at 0.022 g L−1 (䊉) or immobilized HRP ([HRP]ads = 0.022 g L−1 ) onto PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) ()), as a function of time.
408
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
Fig. 5. Enzymatic activity of free HRP at 0.022 g L−1 (white column) and immobilized HRP ([HRP]ads = 0.022 g L−1 ) onto PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) (patterned column), expressed in terms of concentration of radical cation ABTS•+ formed after 10 min of reaction, as a function of storing time.
tions of free HRP and HRP-covered PMMA/CMC particles were stored in closed vials at (24 ± 1) ◦ C for different periods of time and after each period of storing time the catalytic activity of free and immobilized HRP was determined. For the sake of comparison the concentration of free and immobilized HRP was the same and corresponded to that at the adsorption plateau, namely, [HRP]ads = 0.022 g L−1 . Fig. 5 shows the enzymatic activity of free HRP and immobilized HRP, expressed in terms of concentration of radical cation ABTS•+ formed after 10 min of reaction, as a function of storing time. One observes that upon adsorbing HRP, there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. These findings show that (i) storing did not cause changes in the HRP secondary structure and (ii) the surface of PMMA/CMC hybrid particles provided an environment, which prevented HRP conformational changes. Recently [19], measurements of adhesion forces have shown that PMMA/CMC hybrid particles present a hard core and a fluffy layer (20 ± 10) nm thick, which was attributed to the highly hydrated CMC outermost layer around the particle. Similarly HRP molecules presented no reduction of enzymatic activity when they were entrapped in mesoporous silica particles [10], which are very hydrophilic surfaces [30]. Similar behavior for other enzymes can be found in the literature. The catalytic behavior of creatine phosphokinase (CPK) immobilized onto Si/SiO2 wafers under different pH conditions showed that under alkaline conditions, the contribution of strong hydration forces weakened CPK unfolding upon electrostatically driven adsorption [31]. The preservation of enzymatic activity of hexokinase adsorbed onto Si/SiO2 wafers was associated to the interface hydration, which avoided hexokinase denaturation after usage or upon storing [32]. High hydration level on the surface seems to play a very important role, preserving enzyme conformation upon adsorption. Colloidal particles of block copolymer poly(styreneb-acrylic acid), PS-b-PAA, were used for the immobilization of pectinase [33]. The carboxyl groups on the particles surface
Fig. 6. Enzymatic activity of immobilized HRP ([HRP]ads = 0.025 g L−1 ) onto PMMA/CMC particles (Np 2.8 × 1011 particles mL−1 ) freshly prepared () and after drying and re-dispersion () expressed in terms of concentration of radical cation ABTS•+ formed after 10 min of reaction, as a function of time.
led to improved thermal and storage stabilities of immobilized pectinase. Microspheres of PMMA functionalized with acrolein were used for the immobilization of trypsin with successful performance, since increase in thermal stability, pH stability and storage lifetime has been observed [34]. Another interesting result was observed when dispersions of HRP-covered PMMA/CMC particles were dried at 24 ± 1 ◦ C during 72 h and re-dispersed in NaCl 0.001 mol L−1 at the same temperature. The re-dispersed HRP-covered PMMA/CMC particles retained 50% of their original enzymatic activity (Fig. 6), showing that drying brought about conformational changes in HRP structure. Many of water molecules around CMC layers are removed upon drying, perturbing HRP native structure. This finding evidences the crucial role played by surface hydration on the activity of immobilized enzymes. There are interesting reports in the literature [35–37], which show that hydrophobic polystyrene particles are not appropriate for enzyme immobilization. Recently Koutsopoulos et al. [35] observed that upon adsorbing trypsin onto PS films the enzymatic activity was completely suppressed. Caruso et al. [36,37] modified PS latex particles using layer-by-layer polyelectrolyte adsorption technique to facilitate subsequent enzyme adsorption. Four alternating poly(allylamine hydrochloride), PAH, and poly(styrenesulfonate), PSS, layers were deposited onto the PS lattices (first layer PAH), resulting in negatively charged particles with PSS as the outermost layer. The enzyme beta glucosidase [36] or glucose oxidase [37] was then adsorbed onto the outermost PSS layer. The enzyme multilayer-coated particles were enzymatically active, with the general finding that the total activity of the particles increased with increasing enzyme layer number. 4. Conclusions HRP adsorbed irreversibly onto PMMA/CMC hybrid particles, probably due to favorable interaction between HRP carbohydrate residues and CMC segments. HRP-covered PMMA/CMC particles work as catalytic devices with performance comparable to that of free HRP molecules. Such particles can be stored for long terms under room temperature
R.A. Silva et al. / International Journal of Biological Macromolecules 41 (2007) 404–409
without enzymatic activity loss. Moreover, dried HRP-covered PMMA/CMC particles can be added to the desired reaction yielding 50% of original enzymatic activity. Preservation of enzyme activity on PMMA/CMC was attributed to the presence of hydrated CMC layers around the particles. Acknowledgments The authors acknowledge FAPESP and CNPq for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijbiomac.2007.05.014. References [1] I. Chibata, T. Tosa, T. Sato, T. Mori, Immobilized Enzymes, Wiley & Sons, New York, 1978. [2] N.C. Veitch, Phytochemistry 65 (2004) 249–259. [3] G.I. Berglund, G.H. Carlsson, A.T. Smith, H. Sz¨oke, A. Henriksen, J. Hajdu, Nature 417 (2002) 463–468. [4] J.S. Dordick, M.A. Marletta, A.M. Klibanov, Biotechnol. Bioeng. 30 (1987) 31–36. [5] B. Kalra, R.A. Gross, Biomacromolecules 1 (2000) 501–505. [6] B. Kalra, R.A. Gross, Green Chem. 4 (2002) 174–178. [7] W. Liu, J. Kumar, S. Tripathy, K.J. Senecal, L. Samuelson, J. Am. Chem. Soc. 121 (1999) 71–78. [8] J. Shan, Y. Kitamura, H. Yoshizawa, Colloid Polym. Sci. 284 (2005) 108–111. [9] A.F. Naves, A.M. Carmona-Ribeiro, D.F.S. Petri, Langmuir 23 (2007) 1981–1987. [10] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Chem. Mater. 12 (2000) 3301–3305. [11] F. Vianello, L. Zennaro, M.L. Di Paolo, A. Rigo, C. Malacarne, M. Scarpa, Biotechnol. Bioeng. 68 (2000) 488–495. [12] S. Yabuki, F. Mizutami, Y. Hirata, Sens. Actuators B: Chem. 65 (2000) 46–48.
409
[13] G.H. Wang, L.M. Zhang, J. Phys. Chem. B 110 (2006) 24864–24868. [14] N.Q. Jia, J. Xu, H.M. Sun, Z.Y. Jiang, Anal. Lett. 38 (2005) 123–124. [15] L.B.R. Castro, K.V. Soares, A.F. Naves, A.M. Carmona-Ribeiro, D.F.S. Petri, Ind. Eng. Chem. Res. 43 (2004) 7774–7779. [16] A.F. Naves, P.M. Kosaka, J.R. Matos, D.F.S. Petri, J. Therm. Anal. Calorim. 79 (2005) 389–392. [17] A.M. Carmona-Ribeiro, D.F.S. Petri, in: H.S. Nalwa (Ed.), Polymeric Nanostructures and their Applications, vol. 1, American Scientific Publishers, California, 2007, pp. 485–529. [18] K.V. Soares, J.C. Masini, R.M. Torresi, A.M. Carmona-Ribeiro, D.F.S. Petri, Langmuir 21 (2005) 8515–8519. [19] L.B.R. Castro, M. Kappl, D.F.S. Petri, Langmuir 22 (2006) 3757– 3762. [20] L.B.R. Castro, D.F.S. Petri, J. Nanosci. Nanotech. 5 (2005) 2063–2069. [21] K. Chattopadhyay, S. Mazumdar, Biochemistry 39 (2000) 263–270. [22] A.F. Naves, D.F.S. Petri, Colloid Surf. A: Physicochem. Eng. Aspects 254 (2005) 207–214. [23] E.F. Grabowski, I.D. Morrison, in: B. Dahneke (Ed.), Measurements of Suspended Particles by Quasi-Elastic Light Scattering, Wiley–Interscience, New York, 1983 (chapter 7). [24] E.N. Kadnikova, N.M. Kostic, J. Mol. Catal. B: Enzym. 18 (2002) 39–48. [25] R.E. Childs, W.G. Bardsley, Biochem. J. 145 (1975) 93–103. [26] M. Laberge, Q. Huang, R. Schweitzer-Stenner, J. Fidy, Biophys. J. 84 (2003) 2542–2552. [27] http://www.rcsb.org/pdb ID: 1H58, accessed in January 17, 2007. [28] D. Schomberg, M. Salzmann, D. Stephan, Enzyme Handbook, vol. 7, 1993, EC 1.11.1.7:1–6. [29] L.M. Shannon, E. Kay, J.Y. Lew, J. Biol. Chem. 241 (1966) 2166–2168. [30] L.T. Zhuravlev, Colloid Surf. A: Physicochem. Eng. Aspects 173 (2000) 1–38. [31] S.M. Pancera, H. Gliemann, T. Schimmel, D.F.S. Petri, J. Phys. Chem. B 110 (2006) 2674–2680. [32] S.M. Pancera, H. Gliemann, T. Schimmel, D.F.S. Petri, J. Colloid Interf. Sci. 302 (2006) 417–423. [33] Z. Lei, S. Bi, J. Biotechnol. 128 (2007) 112–119. [34] S. Wu, B. Liu, S. Li, Int. J. Biol. Macromol. 37 (2005) 263–267. [35] S. Koutsopoulos, K. Patzsch, W.T.E. Bosker, W. Norde, Langmuir 23 (2007) 2000–2006. [36] F. Caruso, H. Fiedler, K. Haage, Colloid Surf. A: Physicochem. Eng. Aspects 169 (2000) 287–293. [37] F. Caruso, C. Sch¨uler, Langmuir 16 (2000) 9595–9603.