Applied Biochemistry and Biotechnology

, Volume 44, Issue 3, pp 213–229

Contribution to the study of the alteration of lipase activity ofCandida rugosa by ions and buffers

  • M. J. Hernáiz
  • M. Rua
  • B. Celda
  • P. Medina
  • J. V. Sinisterra
  • J. M. Sánchez-Montero


A semipurifiedC. rugosa lipase (LS) has been prepared from commercial lipase (LC) using an economical procedure. The presence of sugars and glycopeptides has been detected in LS and LC. Pure lipase only has covalently bonded sugars. The hydrolysis of olive oil catalyzed by LS and commercial lipase (LC) is sensitive to the presence of cations Na(I), Mg(II), Ca(II), and Ba(II) and to the nature of buffer. Highest enzyme activity is obtained with 0.1M Tris/HCl buffers and the combination of NaCl 0.11M and CaCl2 0.11M. Fluorescence spetroscopy analysis of LC, LS, and both pure isoenzymes lipases A and B, was used to analyze the interaction of the lipase with these effecttors. Inorganic cations Na or Ca do not interact with pure enzyme LA but do interact with LC and LS and do so slightly with LB. The organic cations (morfolinium ortris) interact with pure lipases. We postulate that the increase in the lipase activity produced by Na(I) or Ca(II) is related with interfacial phenomena, but the increase might be more specific in the hydrolysis of olive oil in the presence of Tris-HCl or morfoline-HCl buffer, owing to enzyme-buffer interaction.


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  1. 1.
    Han, D. and Rhee, J. S. (1986),Biotechnol. Bioeng. 28, 1250–1255.CrossRefGoogle Scholar
  2. 2.
    Pronk, W., Boswinkel, G., and Van't Riet, K. (1992),Biocatalysis 5, 305–323.CrossRefGoogle Scholar
  3. 3.
    Goldberg, M., Thomas, D., and Legoy, M. D. (1990),Eur. J. Biochem. 190, 603–609.CrossRefGoogle Scholar
  4. 4.
    Sánchez-Montero, J. M., Hamon, V., Thomas, D., and Legoy, M. D. (1991),Biochim. Biophys. Acta. 1078, 341–350.Google Scholar
  5. 5.
    Brockman, H., Momsen, W. E., and Tsujita, T. (1988),J. A. O. C. S. 65, 891–896.CrossRefGoogle Scholar
  6. 6.
    Ucar, T., Ekiz, H. I., and Caglar, A. (1989),Biotechnol. Bioeng. 33, 1213–1218.CrossRefGoogle Scholar
  7. 7.
    Tsai, S. V., Wu, G. H., and Chiang, C. L. (1991).Biotechnol. Bioeng. 38, 761–766.CrossRefGoogle Scholar
  8. 8.
    Sarda, L. and Desnuelle, P. (1958),Biochim. Biophys. Acta. 30, 513–521.CrossRefGoogle Scholar
  9. 9.
    Verger, R. (1989), inMethods in Enzymology, Purich, D. L., ed., Academic Press, New York, pp. 340–392.Google Scholar
  10. 10.
    White, S. P. (1990).Science 250, 1560–1563.CrossRefGoogle Scholar
  11. 11.
    Brockman, H. L. (1984), inLipases, Borgstron, B. and Brockman, H. L., eds., Elsevier, Amsterdam, The Netherlands, pp. 4–37.Google Scholar
  12. 12.
    Zaks, A. and Klibanov, A. M. (1985),Proc. Natl. Acad. Sci. USA 82, 3192–3196.CrossRefGoogle Scholar
  13. 13.
    Otero, C., Pastor, E., Rua, M., and Ballesteros, A. (1991).Ann. NY Acad. Sci. 523–528.Google Scholar
  14. 14.
    Borgstrom, B. and Erlanson, C. (1973),Eur. J. Biochem. 37, 60–68.CrossRefGoogle Scholar
  15. 15.
    Tanaka, T., Ono, E., Ishihara, M., Yamanaka, S., and Takinani, T. (1981).Agric. Biol. Chem. 45, 2387–2389.Google Scholar
  16. 16.
    Ferreira, G. C. and Paton, J. S. (1990),J. Lipid. Res. 31, 889–897.Google Scholar
  17. 17.
    Rua, M. L. (1992), PhD Thesis. Universidad Autónoma de Madrid.Google Scholar
  18. 18.
    Rua, M. L., Diaz-Maurino, T., Fernandez, V., Otero, C., and Ballesteros, A. (1993)Biochim. Biophys. Acta. 1156, 181–189.Google Scholar
  19. 19.
    Ampon, K., Salleh, A. B., Sallam, F., Wan Yunus, W. M. Z., Razak, C. N. A. and Basri, M. (1991),Enzyme Microb. Technol. 13, 597–601.CrossRefGoogle Scholar
  20. 20.
    Kawaguchi, Y. and Honda, H. (1992), inLipases; Mechanism and Genetic Engineering. Alberghina, L., Schmid, Rd, and Virger, R., eds., VCU, Weinheim, Germany, pp. 221–230.Google Scholar
  21. 21.
    Brady, L. Brzozowski, A. M., Derewenda, Z. S., Dodson, E., Dodson, G., Tolley, S. H., Turkenburg, J. P., Christiansen, L., Huge-Jensen, B., Norskov, L., Thim, L., and Menge, U. (1990),Nature 343, 767–770.CrossRefGoogle Scholar
  22. 22.
    Hartree, E. F. (1972),Anal. Biochem. 48, 422–427.CrossRefGoogle Scholar
  23. 23.
    Edelhoch, H. (1967),Biochemistry 6, 7.Google Scholar
  24. 24.
    The enzymatic activity (μmol trioleine hydrol/min) vs (Enz)-mg/mL was lineal between 0.0–1.8 mg/mL.Google Scholar
  25. 25.
    Lamare, S., Sánchez-Montero, J. M. and Legoy, M. D. (1991),Ann. NY Acad. Sci. 672, 171–177.CrossRefGoogle Scholar
  26. 26.
    Gu, Q. M. and Sih, C. J. (1992),Biocatalysis 6, 115–126.CrossRefGoogle Scholar
  27. 27.
    Arroyo, M., Moreno, J. M., and Sinisterra, J. V. (1992), International Symposium “Stability and Stabilization of Enzymes.” Maastricht, The Netherlands.Google Scholar
  28. 28.
    Shaw, J. F., Chang, R. Ch., Wang, F. F., and Wang, Y. J. (1990),Biotechnol. Bioeng. 35, 132–137.CrossRefGoogle Scholar
  29. 29.
    Longhi, S., Fusetti, F., Grandori, R., Lotti, M., Vanoni, M., and Alberghina, L. (1992).Biochim. Biophys. Acta 1131, 227–232.Google Scholar
  30. 30.
    Kang, S. T. and Rhee, J. Sh. (1989),Biotechnol. Bioeng. 33, 1469–1476.CrossRefGoogle Scholar
  31. 31.
    Stark, M. B. and Holmberg, K. (1989).Biotechnol. Bioeng. 34, 942–950.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1994

Authors and Affiliations

  • M. J. Hernáiz
    • 1
  • M. Rua
    • 2
  • B. Celda
    • 3
  • P. Medina
    • 3
  • J. V. Sinisterra
    • 3
  • J. M. Sánchez-Montero
    • 1
  1. 1.Department of Organic and Pharmaceutical Chemistry, Faculty of PharmacyUniversidad ComplutenseMadridSpain
  2. 2.Instituto de CatálisisCSICMadridSpain
  3. 3.Department of Physical Chemistry, Faculty of ChemistryUniversidad de ValenciaSpain

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