Applied Biochemistry and Biotechnology

, Volume 108, Issue 1–3, pp 787–797 | Cite as

Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems

  • George G. G. Oliveira
  • Daniel P. Silva
  • Ines Conceição Roberto
  • Michele Vitolo
  • Adalberto PessoaJr.
Article

Abstract

This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K Prot ) was 0.47.

Index Entries

Hexokinase liquid-liquid extraction aqueous two-phase systems Saccharomyces cerevisiae 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Kovák, L., Nelson, B. D., and Ernster, L. (1986), Biochem. Biophys. Res. Commum. 134(1), 285–291.CrossRefGoogle Scholar
  2. 2.
    Bergmeyer, H. (1984), Methods of Enzymatic Analysis, 3nd ed., Verlag Chimie, Weinheim, Germany.Google Scholar
  3. 3.
    Chenault, H. K., Mandes, R. F., and Hornberger, K. R. (1997), J. Org. Chem. 62, 331–336.PubMedCrossRefGoogle Scholar
  4. 4.
    Whitaker, J. R. (1991), in Food Enzymology, vol. 2, Fox, P. F., ed., Elsevier, New York, NY.Google Scholar
  5. 5.
    Godfrey, T. and West, S. (1996) Industrial Enzymology, MacMillan, London, England, UK.Google Scholar
  6. 6.
    Rodrigues, E. M. G., Pessoa-Jr., A., and Milagres, A. M. F. (1999), Appl. Biochem. Biotechnol. 78, 779–788.CrossRefGoogle Scholar
  7. 7.
    Rodrigues, E.M.G., Milagres, A.M.F., and Pessoa-Jr., A. (1999), Process Biochem. 34, 121–125.CrossRefGoogle Scholar
  8. 8.
    Diamond, A. D. and Hsu, J. T. (1992), Adv. Biochem. Eng. Biotechnol. 47, 89–135.PubMedGoogle Scholar
  9. 9.
    Costa, S. A., Pessoa Jr., A., and Roberto, I. C. (1998), Appl. Biochem. Biotechnol. 70/72, 629–639.CrossRefGoogle Scholar
  10. 10.
    Gaikar, G. V., Bodhankar, S. S., and Latha, V. J. (1996), J. Chem. Technol. Biotechnol. 67, 329–332.CrossRefGoogle Scholar
  11. 11.
    Coimbra, J. S. R., Thommes, J., Meirelles, A. J. A., and Kula, M. R. (1995), Bioseparation 5, 259–268.Google Scholar
  12. 12.
    Coimbra, J. S. R., Thommes, J., and Kula, M. R. (1994), J. Chromatogr. 668, 85–94.CrossRefGoogle Scholar
  13. 13.
    Coimbra, J. S. R., Mojola, F., and Meirelles, A. J. A. (1998), J. Chem. Eng. Japan 31, 277–285.CrossRefGoogle Scholar
  14. 14.
    Schmidt, A. S., Venton, A. M., and Asenjo, J. A. (1994), Enzyme Microb. Technol. 16, 131–142.CrossRefGoogle Scholar
  15. 15.
    Albertsson, P. A. (1986), Partition of Cell Particles and Macromolecules, John Wiley, New York, NY.Google Scholar
  16. 16.
    Box, G. P., Hunter, W. G., and Hunter, J. S. (1978) Statistics for Experimenters, John Wiley, New York, NY.MATHGoogle Scholar
  17. 17.
    Bradford, M. A. (1976), Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  18. 18.
    Vernau, J. and Kula, M. R. (1990), Biotechnol. Appl. Biochem. 12, 397–404.Google Scholar
  19. 19.
    Geneviève, M. F., Walker, S. G., and Lyddiatt, A. (2000), J. Chromatogr. B 743(1), 409–419.CrossRefGoogle Scholar
  20. 20.
    Cascone, O., Andrews, B. A., and Asenjo, J. A. (1991), Enzyme Microb. Technol. 13, 629–635.CrossRefGoogle Scholar
  21. 21.
    Jain, A and Johri, B. N. (1999), Bioresour. Technol. 67, 205–207.CrossRefGoogle Scholar
  22. 22.
    Morré, D. J., Morré, D. M. (2000), J. Chromatography B 743, 369–376.CrossRefGoogle Scholar
  23. 23.
    Rito-Palomares, M. and Cueto, L. (2000), J. Chromatogr. B 743, 5–12.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • George G. G. Oliveira
    • 1
  • Daniel P. Silva
    • 1
  • Ines Conceição Roberto
    • 2
  • Michele Vitolo
    • 1
  • Adalberto PessoaJr.
    • 1
  1. 1.Department of Biochemical and Pharmaceutical Technology, Faculty of PharmacyUniversity of São PauloSão Paulo, SPBrazil
  2. 2.DEBIQ-FAENQUILLorena, SPBrazil

Personalised recommendations