Applied Biochemistry and Biotechnology

, Volume 169, Issue 3, pp 786–794 | Cite as

Effects of Alcohols and Compatible Solutes on the Activity of β-Galactosidase

  • Andrew N. W. Bell
  • Emma Magill
  • John E. Hallsworth
  • David J. Timson
Article

Abstract

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell’s macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme β-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V max, to fall and the Michaelis constant, K m, to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited β-galactosidase, the addition of compatible solutes was unable to restore the enzyme’s kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme’s structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.

Keywords

Protein denaturation Chaotropic solute Model enzyme Steady-state kinetics Polyol Enzyme flexibility 

Notes

Acknowledgments

JEH acknowledges funding from the Biotechnology and Biological Sciences Research Council (project BBF0034711). AWNB received a postgraduate studentship from the Department of Agriculture and Rural Development Northern Ireland and EM received a Nuffield Foundation Science Bursary.

References

  1. 1.
    Hallsworth, J. E. (1998). Journal of Fermentation and Bioengineering, 85, 125–137.CrossRefGoogle Scholar
  2. 2.
    Hallsworth, J. E., Heim, S., & Timmis, K. N. (2003). Environmental Microbiology, 5, 1270–1280.CrossRefGoogle Scholar
  3. 3.
    Hallsworth, J. E., Prior, B. A., Nomura, Y., Iwahara, M., & Timmis, K. N. (2003). Applied and Environmental Microbiology, 69, 7032–7034.CrossRefGoogle Scholar
  4. 4.
    Bhaganna, P., Volkers, R. J., Bell, A. N., Kluge, K., Timson, D. J., McGrath, J. W., et al. (2010). Microbial Biotechnology, 3, 701–716.CrossRefGoogle Scholar
  5. 5.
    Nicolaou, S. A., Gaida, S. M., & Papoutsakis, E. T. (2010). Metabolic Engineering, 12, 307–331.CrossRefGoogle Scholar
  6. 6.
    Shifrin, S., & Hunn, G. (1969). Archives of Biochemistry and Biophysics, 130, 530–535.CrossRefGoogle Scholar
  7. 7.
    Matsue, S., & Miyawaki, O. (2000). Enzyme MicrobTechnol, 26, 342–347.CrossRefGoogle Scholar
  8. 8.
    Pereira-Rodriguez, A., Fernandez-Leiro, R., Gonzalez-Siso, M. I., Cerdan, M. E., Becerra, M., & Sanz-Aparicio, J. (2012). Journal of Structural Biology, 177, 392–401.CrossRefGoogle Scholar
  9. 9.
    Dickson, R. C., Dickson, L. R., & Markin, J. S. (1979). Journal of Bacteriology, 137, 51–61.Google Scholar
  10. 10.
    Becerra, M., Cerdan, E., & Siso, M. I. G. (1998). Biotechnology Techniques, 12, 253–256.CrossRefGoogle Scholar
  11. 11.
    Kim, C. S., Ji, E. S., & Oh, D. K. (2003). Biotechnology Letters, 25, 1769–1774.CrossRefGoogle Scholar
  12. 12.
    Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). Protein Science. doi: 10.1002/pro.2165.
  13. 13.
    Maksimainen, M., Paavilainen, S., Hakulinen, N., & Rouvinen, J. (2012). FEBS Journal, 279, 1788–1798.CrossRefGoogle Scholar
  14. 14.
    Juers, D. H., Heightman, T. D., Vasella, A., McCarter, J. D., Mackenzie, L., Withers, S. G., et al. (2001). Biochemistry, 40, 14781–14794.CrossRefGoogle Scholar
  15. 15.
    Richard, J. P., Huber, R. E., Heo, C., Amyes, T. L., & Lin, S. (1996). Biochemistry, 35, 12387–12401.CrossRefGoogle Scholar
  16. 16.
    Richard, J. P., Huber, R. E., Lin, S., Heo, C., & Amyes, T. L. (1996). Biochemistry, 35, 12377–12386.CrossRefGoogle Scholar
  17. 17.
    Marquardt, D. (1963). SIAM Journal on Applied Mathematics, 11, 431–441.CrossRefGoogle Scholar
  18. 18.
    Cray, J. A., Russell, J. T., Timson, D. J., Singhal, R. S., & Hallsworth, J. E. (2013). Environmental Microbiology. doi: 10.1111/1462-2920.12018.
  19. 19.
    Hallsworth, J. E., Nomura, Y., & Iwahara, I. (1998). Journal of Fermentation and Bioengineering 86, 451–456.Google Scholar
  20. 20.
    Hallsworth, J. E., Yakimov, M. M., Golyshin, P. N., Gillion, J. L., de D’Auria, G., La Lima Alves, F., et al. (2007). Environmental Microbiology, 9, 801–813.CrossRefGoogle Scholar
  21. 21.
    Sinnott, M. L., & Souchard, I. J. (1973). Biochemical Journal, 133, 89–98.Google Scholar
  22. 22.
    Richard, J. P., Westerfeld, J. G., Lin, S., & Beard, J. (1995). Biochemistry, 34, 11713–11724.CrossRefGoogle Scholar
  23. 23.
    Cornish-Bowden, A. (2004). Fundamentals of enzyme kinetics. London: Portland Press.Google Scholar
  24. 24.
    Liu, G. X., Kong, J., Lu, W. W., Kong, W. T., Tian, H., Tian, X. Y., et al. (2011). Journal of Dairy Science, 94, 5811–5820.CrossRefGoogle Scholar
  25. 25.
    Brown, A. D. (1978). Advances in Microbial Physiology, 17, 181–242.CrossRefGoogle Scholar
  26. 26.
    Goodey, N. M., & Benkovic, S. J. (2008). Nature Chemical Biology, 4, 474–482.CrossRefGoogle Scholar
  27. 27.
    Kristiansson, H., & Timson, D. J. (2011). ChemBioChem, 12, 2081–2087.CrossRefGoogle Scholar
  28. 28.
    Brown, A. D. (1990). Microbial water stress physiology. Chichester: Wiley.Google Scholar
  29. 29.
    Williams, J. P., & Hallsworth, J. E. (2009). Environmental Microbiology, 11, 3292–3308.CrossRefGoogle Scholar
  30. 30.
    Wickson, V. M., & Huber, R. E. (1969). Biochimica et Biophysica Acta, 181, 419–425.CrossRefGoogle Scholar
  31. 31.
    Thomas, K. C., Hynes, S. H., & Ingledew, W. M. (1993). Journal of Industrial Microbiology and Biotechnology, 12, 93–98.CrossRefGoogle Scholar
  32. 32.
    Mansure, J. J., Panek, A. D., Crowe, L. M., & Crowe, J. H. (1994). Biochimica et Biophysica Acta, 1191, 309–316.CrossRefGoogle Scholar
  33. 33.
    Madeira, A., Leitao, L., Soveral, G., Dias, P., Prista, C., Moura, T., et al. (2010). FEMS Yeast Research, 10, 252–258.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Andrew N. W. Bell
    • 1
  • Emma Magill
    • 1
  • John E. Hallsworth
    • 1
  • David J. Timson
    • 1
  1. 1.School of Biological Sciences, Medical Biology CentreQueen’s University BelfastBelfastUK

Personalised recommendations