Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles

doi:10.1016/j.ijbiomac.2007.05.014

International Journal of Biological Macromolecules

Volume 41, Issue 4, 1 October 2007, Pages 404-409

https://doi.org/10.1016/j.ijbiomac.2007.05.014 Get rights and content

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.

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 variety 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 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], [13], [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], [16], [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.

Section snippets

Materials

Methyl methacrylate (MMA, Sigma, USA), cetyltrimethylammonium bromide (CTAB, Aldrich, USA), carboxymethyl cellulose (Sigma, USA, M_v ∼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-6-sulfonic acid) (ABTS, Sigma, USA) were used without further purification. HRP purity, also called Reinheitzahl (Rz), was

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

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 without enzymatic activity loss. Moreover, dried HRP-covered PMMA/CMC particles can be added to the desired reaction yielding 50% of original enzymatic activity.

Acknowledgments

The authors acknowledge FAPESP and CNPq for financial support.

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Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles

Abstract

Introduction

Section snippets

Materials

Adsorption of HRP onto PMMA/CMC particles

Conclusions

Acknowledgments

Phytochemistry

Colloid Surf. A: Physicochem. Eng. Aspects

J. Mol. Catal. B: Enzym.

Biophys. J.

Colloid Surf. A: Physicochem. Eng. Aspects

J. Colloid Interf. Sci.

J. Biotechnol.

Int. J. Biol. Macromol.

Colloid Surf. A: Physicochem. Eng. Aspects

Immobilized Enzymes

Nature

Biotechnol. Bioeng.

Biomacromolecules

Green Chem.

J. Am. Chem. Soc.

Colloid Polym. Sci.

Langmuir

Chem. Mater.