Preparation of magnetic chitosan nanoparticles and immobilization of laccase

Abstract

The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and characterizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows: 10 mL of phosphate buffer (0.1 M, pH 7.0) containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg · mL⁻1 of laccase and 1% (v/v) glutaraldehyde, immobilization temperature of 4°C and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability (480 units · g⁻¹ support) and had good storage stability and operation stability. The K _m of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer (0.1 M, pH 3.0) at 37 °C, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis.

Keywords

laccase / magnetic chitosan nanoparticles / immobilization / enzyme activity / stability

References

[1] Setti L., Giuliani S., Spinozzi G., . Laccase Catalyzed- oxidative Coupling of 3-methyl 2-benzothiazolinone Hydrazone and Methoxyphenols[J]. Enzyme Microb. Tech., 1999, 25: 285-289.

[2] Moeder M., Martin C., Koeller G. Degradation of Hydroxylated Compounds Using Laccase and Horseradish Peroxidase Immobilized on Microporous Polypropylene Hollow Fiber Membranes[J]. J. Membrane Sci., 2004, 245: 183-190.

[3] Osiadacz J., Al-Adhami A. J. H., Bajraszewska D., . On the Use of Trametes Versicolor Laccase for the Conversion of 4-methyl-3-hydroxyanthranilic Acid to Actinocin Chromophore[J]. J. Biotechnol, 1999, 2: 141-149.

[4] Srebotnik E., Hammel K. E. Degradation of Nonphenolic Lignin by the Laccase: 1-hydroxybenzotriazole System[J]. J. Biotechnol, 2000, 1: 9-188.

[5] D’Annibale A., Ricci M., Quaratino D., . Panus Tigrinus Efficiently Removes Phenols, Color and Organic Load from Olive-mill Wastewater[J]. Res. Microbiol., 2004, 155: 596-603.

[6] Mousty C., Vieille L., Cosnier S. Laccase Immobilization in Redox Active Layered Double Hydroxides: a Reagentless Amperometric Biosensor[J]. Biosens. Bioelectron., 2007, 22: 1 733-1 738.

[7] Peralta-Zamora P., Pereira C. M., Tiburtius E. R. L., . Decolorization of Reactive Dyes by Immobilized Laccase[J]. Appl. Catal. B-Environ., 2003, 42: 131-144.

[8] Domínguez A., Gómez J., Lorenzo M., . Enhanced Production of Laccase Activity by Trametes Versicolor Immobilized into Alginate Beads by the Addition of Different Inducers[J]. World J. Microb. Biot., 2007, 23: 367-373.

[9] Timur S., Pazarlioğlu N., Pilloton R., . Thick Film Sensors Based on Laccases from Different Sources Immobilized in Polyaniline Matrix[J]. Sensor. Actuat. B, 2004, 97: 132-136.

[10] Jiang D. S., Long S. Y., Huang J., . Immobilization of Pycnoporus Sanguineus Laccase on Magnetic Chitosan Microspheres[J]. Biochem. Eng. J., 2005, 25: 15-23.

[11] A Einbu. Characterisation of Chitin and a Study of Its Acid-Catalysed Hydrolysis [D]. Thesis of Doctor (Norwegian University). 2007: 11

[12] Altun G. D., Cetinus S. A. Immobilization of Pepsin on Chitosan Beads[J]. Food Chem., 2007, 100: 964-971.

[13] Reddy K. R. C., Kayastha A. M. Improved Stability of Urease upon Coupling to Alkylamine and Arylamine Glass and Its Analytical Use[J]. J. Mol. Catal. B-Enzym., 2006, 38: 104-112.

[14] Huang J., Xiao H. Y., Li B., . Immobilization of Pycnoporus Sanguineus Laccase on Copper Tetra-aminophthalocyanine-Fe₃O₄ Nanoparticle Composite[J]. Biotechnol. Appl. Biochem., 2006, 44: 93-100.

[15] Sahmetlioglu E., Yürük H., Toppare L., . Immobilation of Invertase and Glucose Oxidase in Conducting Copolymers of Thiophene Functionalized Poly(Vinyl Alcohol) with Pyrrole[J]. Reac. Func. Polym., 2006, 66: 365-371.

[16] Çetinus S. A., Öztop H. N. Immobilization of Catalase into Chemically Crosslinked Chitosan Beads[J]. Enzyme Microb. Tech., 2003, 32: 889-894.