Skip to main content
SpringerLink
Log in

Activity Enhancement of Candida antarctica Lipase B by Flexibility Modulation in Helix Region Surrounding the Active Site

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k cat values were shown as dominant factor for the enhancement of catalytic efficiency, k cat/K m, since it was 4.1-fold and 3.8-fold, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Koshland, D. E. (1958). Proceedings of the National Academy of Sciences of the United States of America, 44, 98–104.

    Article  CAS  Google Scholar 

  2. Lonhienne, T., Gerday, C., & Feller, G. (2000). Biochimica et Biophysica-Protein Structure and Molecular Enzymology, 1543, 1–10.

    Article  CAS  Google Scholar 

  3. Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., et al. (2011). Science, 332, 234–238.

    Article  CAS  Google Scholar 

  4. Qian, Z., & Lutz, S. (2005). Journal of the American Chemical Society, 127, 13466–13467.

    Article  CAS  Google Scholar 

  5. Qian, Z., Fields, C. J., & Lutz, S. (2007). Chembiochem : A European Journal of Chemical Biology, 8, 1989–1996.

    Article  CAS  Google Scholar 

  6. Qian, Z., Horton, J. R., Cheng, X., & Lutz, S. (2009). Journal of Molecular Biology, 393, 191–201.

    Article  CAS  Google Scholar 

  7. Trodler, P., & Pleiss, J. (2008). BMC Structural Biology, 8, 9.

    Article  Google Scholar 

  8. Klibanov, A. M. (1997). Trends in Biotechnology, 15, 97–101.

    Article  CAS  Google Scholar 

  9. Broos, J., Visser, A. J. W. G., Engbersen, J. F. J., Verboom, W., van Hoek, A., & Reinhoudt, D. N. (1995). Journal of the American Chemical Society, 117, 12657–12663.

    Article  CAS  Google Scholar 

  10. Clark, S. (2004). Philosophical Transactions of the Royal Society B: Biological Sciences, 359, 1299–1307.

    Article  CAS  Google Scholar 

  11. Hong, S. Y., Jung, E. J., Joo, J. C., & Yoo, Y. J. (2012). Biotechnology and Bioprocess Engineering, 17, 687–692.

    Article  CAS  Google Scholar 

  12. Zheng, L., Baumann, U., & Reymond, J. L. (2004). Nucleic Acids Research, 32, e115.

    Article  Google Scholar 

  13. Trodler, P., Nieveler, J., Rusnak, M., Schmid, R. D., & Pleiss, J. (2008). Journal of Chromatography. A, 1179, 161–167.

    Article  CAS  Google Scholar 

  14. Vriend, G. (1990). Journal of Molecular Graphics, 8, 52.

    Article  CAS  Google Scholar 

  15. Thorpe, M. F., Lei, M., Rader, A. J., Jacobs, D. J., & Kuhn, L. A. (2001). Journal of Molecular Graphics and Modelling, 19, 60–69.

    Article  CAS  Google Scholar 

  16. Radivojac, P., Obradovic, Z., Smith, D. K., Zhu, G., Vucetic, S., Brown, C. J., et al. (2004). Protein Science, 13, 71–80.

    Article  CAS  Google Scholar 

  17. Bhaskaran, R., & Ponnuswamy, P. K. (1988). International Journal of Peptide and Protein Research, 32, 241–255.

    Article  CAS  Google Scholar 

  18. Reetz, M. T., Carballeira, J. D., & Vogel, A. (2006). Angewandte Chemie International Edition, 45, 7745–7751.

    Article  CAS  Google Scholar 

  19. Reetz, M. T., Soni, P., Acevedo, J. P., & Sanchis, J. (2009). Angewandte Chemie International Edition, 48, 8268–8272.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-2009-C1AAA001-2009-0093512).

Author information

Authors and Affiliations

  1. Graduate Program of Bioengineering, Seoul National University, Seoul, 151-742, South Korea

    So Yeon Hong & Young Je Yoo

  2. School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, South Korea

    Young Je Yoo

  3. Bio-Max Institute, Seoul National University, Seoul, 151-742, South Korea

    Young Je Yoo

Authors
  1. So Yeon Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Young Je Yoo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Young Je Yoo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hong, S.Y., Yoo, Y.J. Activity Enhancement of Candida antarctica Lipase B by Flexibility Modulation in Helix Region Surrounding the Active Site. Appl Biochem Biotechnol 170, 925–933 (2013). https://doi.org/10.1007/s12010-013-0237-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-013-0237-8

Keywords

Search

Navigation