Applied Biochemistry and Biotechnology

, Volume 37, Issue 3, pp 283–294 | Cite as

Purification and Characterization of a Milk Clotting Protease fromMucor bacilliformis

  • Liliana B. Areces
  • Mirtha Biscoglio De Jiménez Bonino
  • Marina A. A. Parry
  • Elda R. Fraile
  • Héctor M. Fernández
  • Osvaldo Cascone


An acid protease having milk clotting activity has been isolated fromMucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin. Index Entries:Mucor bacilliformis protease; milk clotting enzyme; acid protease; fungal protease; aspartyl protease.


Apply Biochemistry Proteolytic Activity Wheat Bran Mucor Clotting Activity 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Dalgleish, D. G. (1987), inCheese: Chemistry, Physics and Microbiology, vol. 1 (Fox, P. F., ed.), Elsevier, New York, pp. 63–95.Google Scholar
  2. 2.
    Drohse, H. B. and Foltmann, B. (1989),Biochim. Biophys. Acta 995, 221.Google Scholar
  3. 3.
    Fraile, E. R., Muse, J. O., and Bernardinelli, S. E. (1981),Eur. J. Appl. Microbiol. Biotechnol. 13, 191.CrossRefGoogle Scholar
  4. 4.
    Thakur, M. S., Karanth, N. G., and Nand, K. (1990),Appl. Microbiol. Biotechnol. 32, 409.CrossRefGoogle Scholar
  5. 5.
    Arima, K., Yu, J., and Iwasaki, S. (1970), inMethods of Enzymology, vol.19, Perlman, G. E. and Lorand, L., ed., Academic, New York, p. 446.Google Scholar
  6. 6.
    Food Chemical Codex (1981), Food and Nutrition Board. Division of Biological Sciences. Assemble of Life. Nat. Research Council. III ed. National Academic Press. Washington, D.C.Google Scholar
  7. 7.
    Laemmli, U. K. (1970),Nature 227, 680.CrossRefGoogle Scholar
  8. 8.
    Weber, K. and Osborn, M. (1969),J. Biol. Chem. 244, 4406.Google Scholar
  9. 9.
    Shovers, J. and Fossum, G. (1972),J. Dairy Sci. 55, 1532.Google Scholar
  10. 10.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951),J. Biol. Chem. 193, 265.Google Scholar
  11. 11.
    Gray, W. R. (1972), inMethods in Enzymology, vol.25B, Perlman, G. E. and Lorand, L., eds., Academic, New York, p. 121.Google Scholar
  12. 12.
    Tang, J., Sepulveda, P., Marciniszyn, J., Chen, K. C. S., Huang, W. Y., Tao, N., Liu, D., and Lanier, P. (1973),Proc. Natl. Acad. Sci. USA 70, 3437.CrossRefGoogle Scholar
  13. 13.
    Rickert, W. S. and Elliot, J. R. (1973),Can. J. Biochem. 51, 1638.Google Scholar
  14. 14.
    Davies, D. R. (1990),Ann. Rev. Biophys. Chem. 19, 189.CrossRefGoogle Scholar
  15. 15.
    Blundell, T. L., Jenkins, J. A., Sewell, B. T., Pearl, L. H., Cooper, J. B., Tickle, I. J., Veerapandian, B., and Wood, S. P. (1990),J. Mol. Biol. 211, 919.CrossRefGoogle Scholar
  16. 16.
    Foltmann, B. (1987), inCheese, Chemistry, Physics and Microbiology, General Aspects, vol. 1, Fox, P. F., ed., Elsevier Applied Sciences, London.Google Scholar
  17. 17.
    Yu, J., Tamura, G., and Arima, K. (1969),Biochim. Biophys. Acta 171, 138.Google Scholar
  18. 18.
    Larson, M. K. and Whitaker, J. R. (1970),J. Dairy Sci. 53, 253.CrossRefGoogle Scholar
  19. 19.
    Kobayashi, H., Kusakabe, I., and Murakami, K. (1983),Agric. Biol. Chem. 47, 551.Google Scholar
  20. 20.
    Foltman, B. (1966),Compt. Rend. Triv. Lab. Carlsberg 35, 143.Google Scholar
  21. 21.
    Stenbert, M. Z. (1971),J. Dairy Sci. 54, 159.Google Scholar
  22. 22.
    Somkuti, E. A. and Babel, F. J. (1968),J. Bacteriol. 95, 1407.Google Scholar
  23. 23.
    Gilliland, G. L., Winborne, E. L., Nachman, J., and Wlodawer, A. (1990),Proteins Struct. Funct. Genet. 8, 82.CrossRefGoogle Scholar

Copyright information

© The Humana Press Inc 1993

Authors and Affiliations

  • Liliana B. Areces
    • 1
  • Mirtha Biscoglio De Jiménez Bonino
    • 1
  • Marina A. A. Parry
    • 1
  • Elda R. Fraile
    • 2
  • Héctor M. Fernández
    • 2
  • Osvaldo Cascone
    • 2
  1. 1.Institute de Quimica y Fisicoquimica Biológicas (UBA-CONICET)Universidad de Buenos AiresBuenos AiresArgentina
  2. 2.Cátedra de Microbiologia Industrial y Biotecnologia, Facultad de Farmacia y BioquimicaUniversidad de Buenos AiresBuenos AiresArgentina

Personalised recommendations