Applied Biochemistry and Biotechnology

, Volume 146, Issue 1–3, pp 49–56 | Cite as

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

  • Aline G. Cunha
  • Gloria Fernández-Lorente
  • Juliana V. Bevilaqua
  • Jacqueline Destain
  • Lúcia M. C. Paiva
  • Denise M. G. Freire
  • Roberto Fernández-Lafuente
  • Jose M. Guisán


Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.


Immobilized lipase Y. lipolytica Biocatalysis Glutaraldehyde Hydrophobic supports 



Financial support was gratefully received from PETROBRÁS, FUJB, FAPERJ and CAPES. The authors are also grateful to Prof Rodrigo Volcan Almeida for his contribution.


  1. 1.
    Cammarota, M. C., & Freire, D. M. G. (2006). Bioresource Technology, 97, 2195–2210.CrossRefGoogle Scholar
  2. 2.
    Gotor-Fenandez, V., Brieva, R., & Gotor, V. (2006). Journal Molecular Catalysis B: Enzymatic, 40, 111–120.CrossRefGoogle Scholar
  3. 3.
    Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisán, J. M., & Fernandez-Lafuente, R. (2007). Enzyme and Microbial Technology, 40, 1451–1463.CrossRefGoogle Scholar
  4. 4.
    Koeller, K. M., & Wong, C.-H. (2001). Nature, 409, 232–240.CrossRefGoogle Scholar
  5. 5.
    Bevilaqua, J. B., Lima, L. M., Cunha, A. G., Barreiro, E. J., Alves, T. L. M., & Paiva, L. M. C., et al. (2005). Applied Biochemistry and Biotechnology, 121, 117–128.CrossRefGoogle Scholar
  6. 6.
    Shibatane, T., Omori, K., Akatsuka, H., Kawai, E., & Matsumae, H. (2000). Journal of Molecular Catalysis B: Enzymatic, 10, 141–149.CrossRefGoogle Scholar
  7. 7.
    Mosbach, K. (1971). Science American, 224, 26–32.CrossRefGoogle Scholar
  8. 8.
    Palomo, J. M., Segura, R. L., Fernandez-Lorente, G., Guisán, J. M., & Fernandez-Lafuente, R. (2007). Enzyme and Microbial Technology, 40, 704–707.CrossRefGoogle Scholar
  9. 9.
    Villeneuve, P., Muderhwa, J. M., Graille, J., & Haas, M. J. (2000). Journal of Molecular Catalysis B: enzymatic, 9, 113–148.CrossRefGoogle Scholar
  10. 10.
    Gupta, M. N. (1991). Biotechnology Applied Biochemistry, 14, 1–11.Google Scholar
  11. 11.
    Klibanov, A. M. (1982). Advanced Applied Microbiology, 29, 1–28.CrossRefGoogle Scholar
  12. 12.
    Klibanov, A. M. (1983). Biochemical Society Transactions, 11, 19–20.Google Scholar
  13. 13.
    Mozhaev, V. V., Melik-Nubarov, N. S., Sergeeva, M. V., Sikrnis, V., & Martinek, K. (1990). Biocatalysis, 3, 179–87.CrossRefGoogle Scholar
  14. 14.
    Nanalov, R. J., Kamboure, M. S., & Emanuiloda, E. I. (1993). Biotechnology Applied Biochemistry, 18, 409–416.Google Scholar
  15. 15.
    Jaeger, K. E., Dijkstra, B. W., & Reetz, M. T. (1999). Annual Review of Microbiology, 53, 315–351.CrossRefGoogle Scholar
  16. 16.
    Aloulou, A., Rodriguez, J. A., Fernandez, S., Osterhout, D., Puccinelli, D., & Carriere, F. (2006). Biochimica et Biophisica Acta, 1761, 995–1013.Google Scholar
  17. 17.
    Fernandez-Lafuente, R., Armisén, P., Sabuquillo, P., Fernández-Lorente, G., & Guisán, J. M. (1998). Chemistry and Physics of Lipids, 93, 185–197.CrossRefGoogle Scholar
  18. 18.
    Palomo, J. M., Muñoz, G., Fernández-Lorente, G., Mateo, C., Fernández-Lafuente, R., & Guisán, J. M. (2002). Journal of Molecular Catalysis B: Enzymatic, 19, 279–286.CrossRefGoogle Scholar
  19. 19.
    Lowry, O., Rosenbrough, M., Farr, A., & Randall, R. (1951). Journal of Biological Chemistry, 193, 265–275.Google Scholar
  20. 20.
    Destain, J., Roblain, D., & Thonart, P. (1997). Biotechnology Letters, 19, 105–107.CrossRefGoogle Scholar
  21. 21.
    Cabrera, Z., Palomo, J. M., Fernandez-Lorente, G., Fernandez-Lafuente, R., & Guisan, J. M. (2007). Enzyme and Microbial Technology, 40, 1280–1285.CrossRefGoogle Scholar
  22. 22.
    Fernandez-Lafuente, R., Rosell, C. M., Rodriguez, V., Santana, C., Soler, G., & Batisda, A., et al. (1993). Enzyme and Microbial Technology, 15, 546–550.CrossRefGoogle Scholar
  23. 23.
    Petkar, M., Lali, A., Caimi, P., & Daminati, M. (2006). Journal of Molecular Catalysis B: enzymatic, 9, 83–90.CrossRefGoogle Scholar
  24. 24.
    Palomo, J. M., Segura, R. L., Fernandez-Lorente, G., Pernas, M., Rua, M. L., & Guisán, J. M., et al. (2004). Biotechnology Progress, 20, 630–635.CrossRefGoogle Scholar
  25. 25.
    Oliveira, D., Feihrmann, A. C., Dariva, C., Cunha, A. G., Bevilaqua, J. V., & Destain, J., et al. (2006). Journal of Molecular Catalysis B: Enzymatic, 39, 117–123.CrossRefGoogle Scholar
  26. 26.
    Lipase Engineering Database is available as
  27. 27.
    Wilson, L., Palomo, J. M., Fernández-Lorente, G., Illanes, A., Guisan, J. M., & Fernandez-Lafuente, R. (2006). Enzyme and Microbial Technology, 38, 975–980.CrossRefGoogle Scholar
  28. 28.
    Palomo, J. M., Fuentes, M., Fernández-Lorente, G., Mateo, C., Guisán, J. M., & Fernández-Lafuente, R. (2003). Biomacromolecules, 4, 1–6.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Aline G. Cunha
    • 1
  • Gloria Fernández-Lorente
    • 2
  • Juliana V. Bevilaqua
    • 3
  • Jacqueline Destain
    • 4
  • Lúcia M. C. Paiva
    • 1
  • Denise M. G. Freire
    • 1
  • Roberto Fernández-Lafuente
    • 2
  • Jose M. Guisán
    • 2
  1. 1.Instituto de Química, Universidade Federal do Rio de Janeiro, Centro de Tecnologia (CT)Ilha do FundãoBrazil
  2. 2.Department of BiocatalysisInstitute of CatalysisCantoblancoSpain
  3. 3.Centro de Pesquisa e Desenvolvimento Leopoldo Américo Miguez de Mello (CENPES)PetrobrasBrazil
  4. 4.Centre Wallon de Biologie Industrielle, Faculté universitaire des Sciences agronomiquesGemblouxBelgium

Personalised recommendations