Advertisement

Biotechnology and Bioprocess Engineering

, Volume 18, Issue 6, pp 1090–1100 | Cite as

Catalytic properties of lipases immobilized onto ultrasound-treated chitosan supports

  • Matheus Dorneles de Mello
  • Deborah Cordeiro
  • Lilian Terezinha Costa
  • Cristian Follmer
Research Paper

Abstract

Ultrasound sonication has been utilized to produce fragmentation of chitosan polymer and hence increase the chitosan surface area, making it more accessible to interactions with proteins. In this context, we have investigated the catalytic properties of lipases from different sources immobilized onto ultrasound-treated chitosan (ChiS) pre-activated with glutaraldehyde (ChiS-G). Atomic force microscopy indicated that ChiS-G displays a more cohesive frame without the presence of sheared/fragmented structures when compared with ChiS, which might be attributed to the cross-linking of the polysaccharide chains. The immobilization efficiency onto ChiS-G and ChiS were remarkably higher than using conventional beads. In comparison with the free enzymes, lipases immobilized onto ChiS show a slight increase of apparent Km and decrease of apparent Vmax. On the other hand, immobilization onto ChiS-G resulted in an increase of Vmax, even though a slight increase of Km was also observed. These data suggest that the activation of chitosan with glutaraldehyde has beneficial effects on the activity of the immobilized lipases. In addition, the immobilization of the lipases onto ChiS-G displayed the best reusability results: enzymes retained more than 50% of its initial activity after four reuses, which might be attributed to the covalent attachment of enzyme to activated chitosan. Overall, our findings demonstrate that the immobilization of lipases onto ultrasound-treated chitosan supports is an effective and low-cost procedure for the generation of active immobilized lipase systems, being an interesting alternative to conventional chitosan beads.

Keywords

lipases chitosan ultrasound cross-linking glutaraldehyde 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bruggink, A., A. J. Straathof, and L. A. van der Wielen (2003) A ‘Fine’ chemical industry for life science products: Green solutions to chemical challenges. Adv. Biochem. Eng. Biotechnol. 80: 69–113.Google Scholar
  2. 2.
    Cherry, J. R. and A. L. Fidantsef (2003) Directed evolution of industrial enzymes: An update. Curr. Opin. Biotechnol. 14: 438–443.CrossRefGoogle Scholar
  3. 3.
    Rodrigues, R. C., C. Ortiz, A. Berenguer-Murcia, R. Torres, and R. Fernández-Lafuente (2013) Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 42: 6290–6307.CrossRefGoogle Scholar
  4. 4.
    Garcia-Galan, C., A. Berenguer-Murcia, R. Fernandez-Lafuente, and R. C. Rodrigues (2011) Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal. 353: 2885–2904.CrossRefGoogle Scholar
  5. 5.
    Jaeger, K. E. and T. Eggert (2002) Lipases for biotechnology. Curr. Opin. Biotechnol. 13: 390–397.CrossRefGoogle Scholar
  6. 6.
    Fan, X., X. Niehus, and G. Sandoval (2012) Lipases as biocatalyst for biodiesel production. Methods Mol. Biol. 861: 471–483.CrossRefGoogle Scholar
  7. 7.
    Schmid, R. and R. Verger (1998) Lipases: Interfacial enzymes with attractive applications. Angew. Chemie Int. 37: 1608–1633.Google Scholar
  8. 8.
    Brady, D. and J. Jordaan (2009) Advances in enzyme immobilization. Biotech. Letters 31: 1639–1650.CrossRefGoogle Scholar
  9. 9.
    Ansari, S. A. and Q. Husain (2012) Potential applications of enzymes immobilized on/in nano materials: A review. Biotechnol. Adv. 30: 512–523.CrossRefGoogle Scholar
  10. 10.
    Jegannathan, K. R., S. Abang, D. Poncelet, E. S. Chan, and P. Ravindra (2008) Production of biodiesel using immobilized lipase — a critical review. Crit. Rev. Biotechnol. 28: 253–264.CrossRefGoogle Scholar
  11. 11.
    Krajewska, B., M. Leszko, and W. Zaborska (1990) Urease immobilized on chitosan membrane: Preparation and properties. J. Chem. Tech. Biotech. 48: 337–350.CrossRefGoogle Scholar
  12. 12.
    Pereira, E. B., G. M. Zanin, and H. F. Castro (2003) Immobilization and catalytic properties of lipase on chitosan for hydrolysis and esterification reactions. Braz. J. Chem. Eng. 20: 343–355.CrossRefGoogle Scholar
  13. 13.
    Pavinatto, F. J., L. Caseli, and O. N. Oliveira (2010) Chitosan in nanostructured thin films. Biomacromol. 11: 1897–1908.CrossRefGoogle Scholar
  14. 14.
    Wang, J., G. Zhao, Y. Li, X. Liu, and P. Hou (2013) Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers. Appl. Microbiol. Biotech. 97: 681–692.CrossRefGoogle Scholar
  15. 15.
    Krajewska, B. (2004) Application of chitin- and chitosan-based materials for enzyme immobilizations: A review. Enz. Microb. Technol. 35: 126–139.CrossRefGoogle Scholar
  16. 16.
    Chiou, S. H. and W. T. Wu (2004) Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups. Biomaterials 25: 197–204.CrossRefGoogle Scholar
  17. 17.
    Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.CrossRefGoogle Scholar
  18. 18.
    Prim, N., M. Sánchez, C. Ruiz, F. I. J. Pastor, and P. Diaz (2003) Use of methylumbeliferyl-derivative substrates for lipase activity characterization. J. Mol. Catalys. B: Enz. 22: 339–346.CrossRefGoogle Scholar
  19. 19.
    Roberts, I. M. (1985) Hydrolysis of 4-methylumbelliferyl butyrate: A convenient and sensitive fluorescent assay for lipase activity. Lipids 20: 243–247.CrossRefGoogle Scholar
  20. 20.
    Kasaai, M. R., J. Arul, and G. Charlet (2008) Fragmentation of chitosan by ultrasonic irradiation. Ultrason. Sonochem. 15: 1001–1008.CrossRefGoogle Scholar
  21. 21.
    Kanwal, F., J. J. Liggat, and R. A. Pethrick (2000) Ultrasonic degradation of polystyrene solutions. Polym. Degrad. Stabil. 68: 445–449.CrossRefGoogle Scholar
  22. 22.
    Arroyo, M., J. M. Sánchez-Montero, and J. V. Sinisterra (1999) Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic media. Enz. Microbial. Tech. 24: 3–12.CrossRefGoogle Scholar
  23. 23.
    Reetz, M. T., A. Zonta, and J. Simpelkam (1996) Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials. Biotech. Bioeng. 49: 527–534.CrossRefGoogle Scholar
  24. 24.
    Barbosa, O., R. Torres, C. Ortiz, and R. Fernandez-Lafuente (2012) Versatility of glutaraldehyde to immobilize lipases: Effect of the immobilization protocol on the properties of lipase B from Candida antarctica. Proc. Biochem. 47: 1220–1227.CrossRefGoogle Scholar
  25. 25.
    Betancor, L., F. López-Gallego, A. Hidalgo, N. Alonso-Morales, G. Dellamora-Ortiz, C. Mateo, R. Fernández-Lafuente, and J. M. Guisán (2006) Different mechanisms of protein immobilization on glutaraldehyde activated supports: Effect of support activation and immobilization conditions. Enz. Microbial. Technol. 39: 877–882.CrossRefGoogle Scholar
  26. 26.
    Fernandez-Lafuente, R., P. Armisén, P. Sabuquillo, G. Fernández- Lorente, and J. M. Guisán (1998) Immobilization of lipases by selective adsorption on hydrophobic supports. Chem. Phys. Lipids 93: 185–197.CrossRefGoogle Scholar
  27. 27.
    Verger, R. (1997) ‘Interfacial activation’ of lipases: Facts and artifacts. Trends Biotechnol. 15: 32–38.CrossRefGoogle Scholar
  28. 28.
    Brzozowski, A. M., U. Derewenda, Z. S. Derewenda, G. G. Dodson, D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A. Patkar, and L. Thim (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351: 491–494.CrossRefGoogle Scholar
  29. 29.
    Fernández-Lorente, G., J. M. Palomo, C. Mateo, R. Munilla, C. Ortiz, Z. Cabrera, J. M. Guisán, and R. Fernandez-Lafuente (2006) Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improved performance. Biomacromol. 7: 2610–2615.CrossRefGoogle Scholar
  30. 30.
    Kilinç, A., M. Teke, S. Onal, and A. Telefoncu (2006) Immobilization of pancreatic lipase on chitin and chitosan. Prep. Biochem. Biotech. 36: 153–163.CrossRefGoogle Scholar
  31. 31.
    Mateo, C., J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan, and R. Fernandez-Lafuente (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enz. Microbial. Technol. 40: 1451–1463.CrossRefGoogle Scholar
  32. 32.
    Bai, Y. X., Y. F. Li, Y. Yang, and L. X. Yi (2006) Covalent immobilization of triacylglycerol lipase onto functionalized novel mesoporous silica supports. J. Biotech. 125: 574–582.CrossRefGoogle Scholar
  33. 33.
    Chen, H. and Y. L. Hsieh (2004) Enzyme immobilization on ultrafine cellulosefibers via poly(acrylic acid) electrolyte grafts. Biotech. Bioeng. 90: 405–413.CrossRefGoogle Scholar
  34. 34.
    Silva, J. E. S. and P. C. Jesus (2003) Evaluation of the catalytic activity of lipases immobilized on chrysotile for esterification. Ann. Acad. Braz. Sci. 75: 157–162.Google Scholar
  35. 35.
    Lee, D. H., C. H. Park, J. M. Yeo, and S. W. Kim (2006) Lipase immobilization on silica gel using a cross-linking method. J. Ind. Eng. Chem. 12: 777–782.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Matheus Dorneles de Mello
    • 1
  • Deborah Cordeiro
    • 1
  • Lilian Terezinha Costa
    • 2
    • 3
  • Cristian Follmer
    • 1
  1. 1.Department of Physical Chemistry, Institute of ChemistryFederal University of Rio de JaneiroRio de JaneiroBrazil
  2. 2.National Institute of MetrologyStandardization and Industrial QualityDuque de CaxiasBrazil
  3. 3.Institute of BiophysicsFederal University of Rio de Janeiro/XerémDuque de CaxiasBrazil

Personalised recommendations