Magnetic Nanoparticles Functionalized And Modified With Cross-Linking To Improve The Invertase Immobilization
- Annie Y. Vargas ,
- Gustavo P. Romanelli ,
- Jose J. Martinez
Abstract
Procedures of immobilization invertase have been developed using different supports. However, disadvantages such as use of small particles for invertase immobilizations in packed-bed reactors are being solved using magnetic particles. In this study, composites containing Fe3O4 were prepared by incorporation of a polysiloxane layer required for the physical adsorption of the invertase. Besides, the functionalized magnetite was activated with glutaraldehyde and polyethylenimine (PEI) with the aim of performing a covalent immobilization. The effect of different conditions such as enzyme:support ratio, pH, and temperature were analyzed in the preservation of invertase. The results demonstrated that the optimum enzyme:support ratio is higher for covalent bonding than for physical adsorption. The ideal pH for the immobilized enzyme is 5.0, and the enzymatic activity is retained until 70°C. The values of km are similar in both immobilization methods. The analysis of the effect of pH and thermostability showed that the catalytic activity of invertase is not affected in comparison with the free enzyme. The covalent immobilization displays higher efficiency in the immobilization process (Fε), less inhibition and twice as much stability. The enzymes immobilized by physical and covalent methods can be reused for up to four cycles and can be removed from the reaction medium by applying an external magnetic field.
Keywords
invertase, Fe3O4, immobilization, composites.
References
[1] S. M. Kotwal and V. Shankar, “Immobilized invertase,” Biotechnol. Adv., vol. 27, no. 4, p. 311–322, 2009.
[2] E. J. Tomotani and M. Vitolo, “Production of high-fructose syrup using immobilized invertase in a membrane reactor,” J. Food Eng., vol. 80, p. 662–667, 2007.
[3] G. Sanjay and S. Sugunan, “Invertase immobilised on montmorillonite : reusability enhancement and reduction in leaching,” Catal. Commun., vol. 6, p. 81–86, 2005.
[4] G. Bayramoglu, M. Karakisla, B. Altıntas, A. Metin, M. Sacak, and M. Y. Arica, “Polyaniline grafted polyacylonitrile conductive composite fibers for reversible immobilization of enzymes : Stability and catalytic properties of invertase,” Process Biochem., vol. 44, p. 880–885, 2009.
[5] M. Azodi, C. Falamaki, and A. Mohsenifar, “Journal of Molecular Catalysis B : Enzymatic Sucrose hydrolysis by invertase immobilized on functionalized porous silicon,” "Journal Mol. Catal. B, Enzym., vol. 69, no. 3–4, p. 154–160, 2011.
[6] L. Amaya-Delgado, M. . Hidalgo-Lara, and M. . Montes-Horcasitas, “Food Chemistry Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads,” Food Chem., vol. 99, p. 299–304, 2006.
[7] S. G. Valerio, J. S. Alves, M. P. Klein, R. C. Rodrigues, and P. F. Hertz, “High operational stability of invertase from Saccharomyces cerevisiae immobilized on chitosan nanoparticles,” Carbohydr. Polym., vol. 92, no. 1, p. 462–468, 2013.
[8] J. A. Solís-Fuentes, E. Raga-Carbajal, and M. C. Durán-de-Bazúa, “Direct Sucrose Hydrolysis in Sugarcane Juice with Immobilized Invertase: Multi-response Optimization Using Desirability Function on Conversion and Reactor Volumetric Productivity,” Sugar Tech, vol. 17, no. 3, p. 266–275, 2015.
[9] R. Mouelhi, F. Abidi, S. Galai, and M. N. Marzouki, “Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by product,” World J. Microbiol. Biotechnol., vol. 30, no. 3, p. 1063–1073, 2014.
[10] Y. Chen, E. T. Kang, K. G. Neoh, and K. L. Tan, “Covalent immobilization of invertase onto the surface- modified polyaniline from graft copolymerization with acrylic acid,” Eur. Polym. J., vol. 36, p. 2095–2103, 2000.
[11] L. Raj, G. S. Chauhan, W. Azmi, J. H. Ahn, and J. Manuel, “Kinetics study of invertase covalently linked to a new functional nanogel,” Bioresour. Technol., vol. 102, no. 3, p. 2177–2184, 2011.
[12] S. Akgöl, Y. Kacar, A. Denizli, and M. Y. Arica, “Hydrolysis of sucrose by invertase immobilized onto novel magnetic polyvinylalcohol microspheres,” Food Chem., vol. 74, p. 281–288, 2001.
[13] P. P. Waifalkar, S. B. Parit, A. D. Chougale, S. C. Sahoo, P. S. Patil, and P. B. Patil, “Immobilization of invertase on chitosan coated γ-Fe2O3 magnetic nanoparticles to facilitate magnetic separation,” J. Colloid Interface Sci., vol. 482, p. 159–164, 2016.
[14] M. P. Cabrera, C. R. D. Assis, D. F. M. Neri, C. F. Pereira, F. Soria, and L. B. Carvalho, “High sucrolytic activity by invertase immobilized onto magnetic diatomaceous earth nanoparticles,” Biotechnol. Reports, vol. 14, p. 38–46, 2017.
[15] G. Bayramoglu, T. Doz, V. C. Ozalp, and M. Y. Arica, “Improvement stability and performance of invertase via immobilization on to silanized and polymer brush grafted magnetic nanoparticles,” Food Chem., vol. 221, p. 1442–1450, 2016.
[16] K. Uzun et al., “Covalent immobilization of invertase on PAMAM- dendrimer modified superparamagnetic iron oxide nanoparticles,” J. Nanopart. Res, vol. 12, p. 3057–3067, 2010.
[17] O. Barbosa, R. Torres, C. Ortiz, N. Berenguer-Murcia, R. C. Rodrigues, and R. Fernandez-Lafuente, “Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties,” Biomacromolecules, vol. 14, no. 8, p. 2433–2462, 2013.
[18] A. Escobar, L. R. Pizzio, and G. P. Romanelli, “Catalizadores magnéticos basados en Óxidos de Hierro: Síntesis, Propiedades y Aplicaciones,” Ciencia En Desarrollo, vol. 10, no. 1, p. 79–101, Dec. 2018.
[19] A. Y. Vargas, H. A. Rojas, G. P. Romanelli, and J. J. Martínez, “Synthesis of 1,4-dihydropyrimidines with immobilized urease: Effect of method immobilization on magnetic supports,” Green Process. Synth., vol. 6, no. 4, p. 377–384, 2017.
[20] Y. S. Kang, S. Risbud, J. F. Rabolt, and P. Stroeve, “Synthesis and Characterization of Nanometer-Size Fe3O4 and γ-Fe2O3 Particles,” Chem. Mater., vol. 8, no. 9, p. 2209–2211, 1996.
[21] B. Luo et al., “Multi-functional thermosensitive composite microspheres with high magnetic susceptibility based on magnetite colloidal nanoparticle clusters,” Langmuir, vol. 26, no. 3, p. 1674–1679, 2010.
[22] P. Díez, R. Villalonga, M. L. Villalonga, and J. M. Pingarrón, “Supramolecular immobilization of redox enzymes on cyclodextrin-coated magnetic nanoparticles for biosensing applications,” J. Colloid Interface Sci., vol. 386, no. 1, p. 181–188, 2012.
[23] O. Lowry, J. Rosebrough, L. Farr, and R. . Randall, “Protein measurement with the Folin phenol reagent,” J. Biol. Chem., vol. 193, no. 1, p. 265–275, 1951.
[24] G. L. Miller, “Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar,” Anal. Chem., vol. 31, no. 3, p. 426–428, 1959.
[25] H. A. Rojas, J. J. Martínez, and A. Y. Vargas, “Selección de soportes magnéticos para la inmovilización de Ureasa Magnetic supports selection for Urease inmobilization,” Ing. y Compet., vol. 296, no. 2, p. 289–296, 2014.
[26] N. Dizge, O. Gunaydin, F. Yilmaz, and A. Tanriseven, “Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix,” Biochem. Eng. J., vol. 40, no. 1, p. 64–71, 2008.