Skip to main content

Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

Abstract

Several micro-scale chromatography-based procedures for purification of the β-galactosidase from the yeast Kluyveromyces lactis were assayed. Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production. Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme.

References

  1. 1.

    González Siso, M.I. 1996. The biotechnological utilization of cheese whey: a review. Biores.Technol. 57, 1–11.

    Article  Google Scholar 

  2. 2.

    González Siso, M.I. 1994. β-galactosidase production by Kluyveromyces lactis on milk whey: batch versus fed-batch cultures. Proc. Biochem. 29, 565–568

    Article  Google Scholar 

  3. 3.

    Becerra, M. and González Siso, M.I. 1996. Yeast β-galactosidase in solid-state fermentations. Enz. Microb.Technol. 19, 33–44.

    Google Scholar 

  4. 4.

    González Siso, M.I., Freire Picos, A., Ramil, E., RodrÍguez Belmonte, E., RodrÍguez Torres, A. and Cerdán, E. 1994. Covalent immobilization of β-galactosidase on corn grits. A system for lactose hydrolysis without diffusional resistance. Proc. Biochem. 29, 7–12

    Article  Google Scholar 

  5. 5.

    González Siso, M.I., Cerdán, E., Freire Picos, M. A., RodrÍguez Belmonte, E. and RodrÍguez Torres, A.M. 1992. Permeabilization of Kluyveromyces lactis cells for milk whey saccharification: a comparison of different treatments. Biotechnol.Techniques 6, 289–292.

    Article  Google Scholar 

  6. 6.

    González Siso, M.I. and Suárez Doval, S. 1994. Kluyveromyces lactis immobilization on corn grits for milk whey lactose hydrolysis. Enzyme Microb. Technol. 16, 303–310.

    Article  Google Scholar 

  7. 7.

    Becerra, M., Cerdán, E. and González Siso, M.I. 1997. Heterologous Kluyveromyces lactis β-galactosidase production and release by Saccharomyces cerevisiae osmotic-remedial thermosensitive autolytic mutants. Biochim. Biophys. Acta. 1335, 235–241.

    PubMed  CAS  Google Scholar 

  8. 8.

    Becerra, M., Cerdán, E. and González Siso, M.I. 1998. Micro-scale purification of β-galactosidase from Kluyveromyces lactis reveals that dimeric and tetrameric forms are active. Biotechnol.Techniques 12, 253–256.

    Article  CAS  Google Scholar 

  9. 9.

    Nader de Macias, M.E., Perdigon, G., Oliver, G. and A. A. P. de Ruiz Holgado. 1985. Immunological relationships among β-galactosidases in members of the genus Lactobacillus. Int. J. Sys. Bacteriol. 35, 103–110.

    CAS  Article  Google Scholar 

  10. 10.

    Guarante, L. 1983. Yeast promoters and LacZ fusions designed to study expression of cloned genes in yeasts. Meth. Enzymol. 101, 181–189.

    Article  Google Scholar 

  11. 11.

    Inchaurrondo, V. A., Yantorno, O.M. and Voget, C.E. 1994. Yeast growh and β-galactosidase production during aerobic batch cultures in lactose limited synthetic medium. Proc. Biochem. 29, 47–54.

    Article  CAS  Google Scholar 

  12. 12.

    Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Neuhoff, V., Stamm, R. and Eibk, M. 1985. Clear background and highly sensitive protein staining with Coomassie blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6, 427–448.

    Article  CAS  Google Scholar 

  15. 15.

    Dickson, R.C., Dickson, L.R. and Markin, J.S. 1979. Purification and properties of an inducible β-galactosidase isolated from the yeast Kluyveromyces lactis. J. Bacteriol. 137, 51–61.

    PubMed  CAS  Google Scholar 

  16. 16.

    Erickson, R.P. and Steers, J.R. E. 1970. Comparative study of isoenzyme formation of bacterial β-Galactosidase. J. Bacteriol. 102, 79–84.

    PubMed  CAS  Google Scholar 

  17. 17.

    Brady, D., Marchant, R., McHale, L. and McHale, A.P. 1995. Isolation and partial characterization of β-galactosidase activity produced by a thermotolerant strain of Kluyveromyces marxianus during growth on lactose-containing media. Enzyme Microb. Technol. 17, 696–699.

    Article  CAS  Google Scholar 

  18. 18.

    Pritchard, D.I., Maizels, R.M., Behnke, J.M. and Appleby, P. 1984. Stage-specific antigens of Nematospiroides dubius. Immunology 53, 325–335.

    PubMed  CAS  Google Scholar 

  19. 19.

    Iglesias, R., Leiro, J., Ubeira, F.M., Santamarina, M.T. and Sanmartín, M.L. 1993. Anisakis simplex: antigen recognition and antibody production in experimentally infected mice. Parasite Immunol. 15, 243–250.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to M. I. González Siso.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Becerra, M., Cerdán, E. & González Siso, M.I. Dealing with different methods for Kluyveromyces lactis β-galactosidase purification. Biol Proced Online 1, 48–58 (1998). https://doi.org/10.1251/bpo4

Download citation

Keywords

  • Lactase
  • Cheese Whey
  • Purification Factor
  • Biological Procedure
  • Crude Protein Extract