Applied Biochemistry and Biotechnology

, Volume 118, Issue 1–3, pp 11–20 | Cite as

Reductive alkylation of lipase

Experimental and molecular modeling approaches
  • Raja Noor Zaliha A. Rahman
  • Bimo Ario Tejo
  • Mahiran Basri
  • Mohd Basyaruddin A. Rahman
  • Farid Khan
  • Sharifuddin M. Zain
  • Teruna J. Siahaan
  • Abu Bakar Salleh
Article

Abstract

Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.

Index Entries

Lipase Candida rugosa enzyme modification circular dichroism molecular modeling 

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References

  1. 1.
    Koskinen, A. M. P. and Klibanov, A. M. (1996), Enzymatic Reactions in Organic Media, Blackie, London.Google Scholar
  2. 2.
    Klibanov, A. M. (1997), Trends Biochem. Sci. 15, 97–101.Google Scholar
  3. 3.
    Ampon, K., Salleh, A. B., Basri, M., Yunus, W. M. Z., Razak, C. N. A., and Whitaker, J. R. (1993), J. Biosci. 4(2), 154–160.Google Scholar
  4. 4.
    Longo, M. A. and Combes, D. (1999), J. Chem. Technol. Biotechnol. 74, 25–32.CrossRefADSGoogle Scholar
  5. 5.
    Inada, Y., Furukawa, M., Sasaki, H., Kodera, Y., Hiroto, M., Nishimura, H., and Matsushima, A. (1986), TIBTECH 13, 86–91.Google Scholar
  6. 6.
    Means, G. E. (1977), in Methods in Enzymology, vol. XLVII, Hirs, C. H. W. and Timasheff, S. N., eds., Academic, New York, pp. 469–478.Google Scholar
  7. 7.
    Grochulski, P., Li, Y., Schrag, J. D., and Cygler, M. (1994), Protein Sci. 3, 82–91.PubMedCrossRefGoogle Scholar
  8. 8.
    Wu, S. H., Guo, Z. W., and Sih, C. J. (1990), J. Am. Chem. Soc. 112, 1990–1995.CrossRefGoogle Scholar
  9. 9.
    Salleh, A. B., Ampon, K., Salam, F., Wan Yunus, W. M. Z., Razak, C. N. A., and Basri, M. (1990), Ann. NY Acad. Sci. 613, 521, 522.CrossRefGoogle Scholar
  10. 10.
    Ampon, K., Salleh, A. B., Salam, F., Wan Yunus, W. M. Z., Razak, C. N. A., and Basri, M. (1991), Enzyme Microb. Technol. 13, 597–601.CrossRefGoogle Scholar
  11. 11.
    Basri, M., Th’ng, B. L., Razak, C. N. A., and Salleh, A. B. (1998), Ann. NY Acad. Sci. 864, 192–197.PubMedCrossRefGoogle Scholar
  12. 12.
    Rua, M. L., Diaz-Maurino, T., Fernandez, V. M., Otero, C., and Ballesteros, A. (1993), Biochim. Biophys. Acta 1156, 181–189.PubMedGoogle Scholar
  13. 13.
    Habeeb, A. F. S. A. (1966), Anal. Biochem. 14, 328–336.PubMedCrossRefGoogle Scholar
  14. 14.
    Sreerama, N. and Woody, R. W. (2000), Anal. Biochem. 282, 252–260.CrossRefGoogle Scholar
  15. 15.
    Andrade, M. A., Chacon, P., Merelo, J. J., and Moran, F. (1993), Protein Eng. 6, 383–390.PubMedCrossRefGoogle Scholar
  16. 16.
    Manavalan, P. and Johnson, W. C., Jr. (1983), Nature 305, 831–832.CrossRefADSGoogle Scholar
  17. 17.
    Secundo, F. and Carrea, G. (2002), J. Mol. Catal. B 19–20, 93–102.CrossRefGoogle Scholar
  18. 18.
    Altamirano, M. M., Blackburn, J. M., Aguayo, C., and Fersht, A. R. (2000), Nature 403, 617–622.PubMedCrossRefADSGoogle Scholar
  19. 19.
    Kumar, S., Wolfson, H., and Nussinov, R. (2001), IBM J. Res. Dev. 45(3/4), 499–512.CrossRefGoogle Scholar
  20. 20.
    Richardson, J. S. and Richardson, D. C. (1988), Science 240, 1648–1652.PubMedCrossRefADSGoogle Scholar
  21. 21.
    Guo, Y. and Clark, D. S. (2001), Biochim. Biophys. Acta 1546, 406–411.PubMedGoogle Scholar
  22. 22.
    Colombo, G., Ottolina, G., and Carrea, G. (2000), Monat. Chem. 131, 527–547.Google Scholar
  23. 23.
    Ma, B., Wolfson, H., and Nussinov, R. (2001), Curr. Opin. Struct. Biol. 11, 364–369.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Raja Noor Zaliha A. Rahman
    • 1
    • 2
  • Bimo Ario Tejo
    • 1
  • Mahiran Basri
    • 1
    • 2
  • Mohd Basyaruddin A. Rahman
    • 2
  • Farid Khan
    • 3
  • Sharifuddin M. Zain
    • 4
  • Teruna J. Siahaan
    • 5
  • Abu Bakar Salleh
    • 1
    • 2
  1. 1.Enzyme and Microbial Technology Research, Faculty of Science and Environmental StudiesUniversiti Putra MalaysiaSerdangMalaysia
  2. 2.Laboratory of Enzyme and Microbial Technology, Institute of BioscienceUniversiti Putra MalaysiaSerdangMalaysia
  3. 3.Centre for Protein Engineering, Department of ChemistryCambridge UniversityCambridgeUK
  4. 4.Department of Chemistry, Faculty of ScienceUniversiti MalayaKuala LumpurMalaysia
  5. 5.Department of Pharmaceutical ChemistryUniversity of KansasLawrence

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