Advertisement

Applied Biochemistry and Biotechnology

, Volume 168, Issue 5, pp 1288–1301 | Cite as

Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers

  • Olivera Prodanović
  • Miloš Prokopijević
  • Dragica Spasojević
  • Željko Stojanović
  • Ksenija Radotić
  • Zorica D. Knežević-Jugović
  • Radivoje Prodanović
Article

Abstract

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g−1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.

Keywords

Macroporous polymers Copolymerization Enzymes Horseradish peroxidase Covalent immobilization Morphology Biological applications of polymers 

Notes

Acknowledgments

This work was supported by Grant No. ON173017 and Grant No. ON172049, sponsored by the Ministry of Education and Science, Republic of Serbia.

References

  1. 1.
    Hernandez, K., Garcia-Galan, C., & Fernandez-Lafuente, R. (2011). Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads. Enzyme and Microbial Technology, 49, 72–78.CrossRefGoogle Scholar
  2. 2.
    Knezevic, Z., Milosavic, N., Bezbradica, D., Jakovljevic, Z., & Prodanovic, R. (2006). Immobilization of lipase from Candida rugosa on Eupergit (R) supports by covalent attachment. Biochemical Engineering Journal, 30, 269–278.CrossRefGoogle Scholar
  3. 3.
    Prlainović, N., Knežević-Jugović, Z., Mijin, D., & Bezbradica, D. (2011). Immobilization of lipase from Candida rugosa on Sepabeads®: The effect of lipase oxidation by periodates. Bioprocess and Biosystems Engineering, 34, 803–810.CrossRefGoogle Scholar
  4. 4.
    Prodanović, R. M., Milosavić, N. B., Jovanović, S. M., Ćirković Veličković, T., Vujčić, Z. M., & Jankov, R. M. (2006). Stabilization of alpha-glucosidase in organic solvents by immobilization on macroporous poly(GMA-co-EGDMA) with different surface characteristics. Journal of the Serbian Chemical Society, 71, 339–347.CrossRefGoogle Scholar
  5. 5.
    Jovanovic, S. M., Nastasovic, A., Jovanovic, N. N., & Jeremic, K. (1996). Targeted porous structure of macroporous copolymers based on glycidyl methacrylate. Materials Science Forum, 214, 155–162.CrossRefGoogle Scholar
  6. 6.
    Klibanov, A. M., Berman, Z., & Alberti, B. N. (1981). Preparative hydroxylation of aromatic compounds catalyzed by peroxidase. Journal of the American Chemical Society, 103, 6263–6264.CrossRefGoogle Scholar
  7. 7.
    Schwartz, R. D., & Hutchinson, D. B. (1981). Microbial and enzymatic production of 4,4′-dihydroxybiphenyl via phenol coupling. Enzyme and Microbial Technology, 3, 361–363.CrossRefGoogle Scholar
  8. 8.
    Klibanov, A. M., Tu, T. M., & Scott, K. P. (1983). Peroxidase-catalyzed removal of phenols from coal-conversion waste waters. Science, 221, 259–261.CrossRefGoogle Scholar
  9. 9.
    Alonso-Lomillo, M. A., Dominguez-Renedo, O., del Torno-de Roman, L., & Arcos-Martinez, M. J. A. (2011). Horseradish peroxidase-screen printed biosensors for determination of Ochratoxin A. Analytica Chimica Acta, 688, 49–53.CrossRefGoogle Scholar
  10. 10.
    Presnova, G. V., Rybcova, M. Y., & Egorov, A. M. (2008). Electrochemical biosensors based on horseradish peroxidase. Russian Journal of General Chemistry, 78, 2482–2488.CrossRefGoogle Scholar
  11. 11.
    Yu, D. H., Blankert, B., & Kauffmann, J. M. (2007). Development of amperometric horseradish peroxidase based biosensors for clozapine and for the screening of thiol compounds. Biosensors & Bioelectronics, 22, 2707–2711.CrossRefGoogle Scholar
  12. 12.
    Guschin, D. A., Sultanov, Y. M., Sharif-Zade, N. F., Aliyev, E. H., Efendiev, A. A., & Schuhmann, W. (2006). Redox polymer-based reagentless horseradish peroxidase biosensors influence of the molecular structure of the polymer. Electrochimica Acta, 51, 5137–5142.CrossRefGoogle Scholar
  13. 13.
    Bayramoglu, M., & Arica, M. Y. (2008). Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: Horseradish peroxidase immobilized on magnetic beads. Journal of Hazardous Materials, 156, 148–155.CrossRefGoogle Scholar
  14. 14.
    Pramparo, L., Stuber, F., Font, J., Fortuny, A., Fabregat, A., & Bengoa, C. (2010). Immobilisation of horseradish peroxidase on Eupergit (R) C for the enzymatic elimination of phenol. Journal of Hazardous Materials, 177, 990–1000.CrossRefGoogle Scholar
  15. 15.
    Monier, M., Ayad, D. M., Wei, Y., & Sarhan, A. A. (2010). Immobilization of horseradish peroxidase on modified chitosan beads. International Journal of Biological Macromolecules, 46, 324–330.CrossRefGoogle Scholar
  16. 16.
    Shukla, S. P., Modi, K., Ghosh, P. K., & Devi, S. (2004). Immobilization of horseradish peroxidase by entrapment in natural polysaccharide. Journal of Applied Polymer Science, 91, 2063–2071.CrossRefGoogle Scholar
  17. 17.
    Alemzadeh, I., & Nejati, S. (2009). Phenols removal by immobilized horseradish peroxidase. Journal of Hazardous Materials, 166, 1082–1086.CrossRefGoogle Scholar
  18. 18.
    Ozoner, S. K. (2012). Poly(glycidyl methacrylate-co-3-thienyl-methylmethacrylate) based working electrodes for hydrogen peroxide biosensing. Journal of Chemical Technology & Biotechnology, 87, 146–152.CrossRefGoogle Scholar
  19. 19.
    Arıca, M. Y., Altıntas, B., & Bayramoglu, G. (2009). Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: Application for textile dye degradation. Bioresource Technology, 100, 665–669.CrossRefGoogle Scholar
  20. 20.
    Prodanovic, R., Jovanovic, S., & Vujcic, Z. (2001). Immobilization of invertase on a new type of macroporous glycidyl methacrylate. Biotechnology Letters, 23, 1171–1174.CrossRefGoogle Scholar
  21. 21.
    Milosavic, N., Prodanovic, R., Jovanovic, S., & Vujcic, Z. (2007). Immobilization of glucoamylase via its carbohydrate moiety on macroporous poly(GMA-co-EGDMA). Enzyme and Microbial Technology, 40, 1422–1426.CrossRefGoogle Scholar
  22. 22.
    Ferreira, L., Ramos, M. A., Dordick, J. S., & Gil, M. H. (2003). Influence of different silica derivatives in the immobilization and stabilization of a Bacillus licheniformis protease (Subtilisin Carlsberg). Journal of Molecular Catalysis B: Enzymatic, 21, 189–199.CrossRefGoogle Scholar
  23. 23.
    Boller, T., Meier, C., & Menzler, S. (2002). Eupergit oxirane acrylic beads: How to make enzymes fit for biocatalysis. Organic Process Research and Development, 6, 509–519.CrossRefGoogle Scholar
  24. 24.
    Torres-Salas, P., Monte-Martinez, A., Cutino-Avila, B., Rodriguez-Colinas, B., Alcalde, A., Ballesteros, A. O., & Plou, F. J. (2011). Immobilized biocatalysts: Novel approaches and tools for binding enzymes to supports. Advanced Materials, 23, 5275–5282.CrossRefGoogle Scholar
  25. 25.
    Garcia, D., Ortega, F., & Marty, J.-L. (1998). Kinetics of thermal inactivation of horseradish peroxidase: Stabilizing effect of methoxypoly(ethylene glycol). Biotechnology and Applied Biochemistry, 27, 49–54.Google Scholar
  26. 26.
    Prodanovic, R., Milosavic, N., Jovanovic, S., Prodanovic, O., Cirkovic Velickovic, T., Vujcic, Z., & Jankov, M. R. (2006). Activity and stability of soluble and immobilized alpha-glucosidase from baker's yeast in cosolvent systems. Biocatalysis and Biotransformation, 24, 195–200.CrossRefGoogle Scholar
  27. 27.
    Bindhu, L. V., & Abraham, E. T. J. (2003). Immobilization of horseradish peroxidase on chitosan for use in nonaqueous media. Journal of Applied Polymer Science, 88, 1456–1464.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Olivera Prodanović
    • 1
  • Miloš Prokopijević
    • 1
  • Dragica Spasojević
    • 1
  • Željko Stojanović
    • 2
  • Ksenija Radotić
    • 1
  • Zorica D. Knežević-Jugović
    • 3
  • Radivoje Prodanović
    • 4
  1. 1.Institute for Multidisciplinary ResearchUniversity of BelgradeBelgradeSerbia
  2. 2.Institute of Chemistry, Technology and MetallurgyUniversity of BelgradeBelgradeSerbia
  3. 3.Faculty of Technology and MetallurgyUniversity of BelgradeBelgradeSerbia
  4. 4.Faculty of ChemistryUniversity of BelgradeBelgradeSerbia

Personalised recommendations