Applied Biochemistry and Biotechnology

, Volume 117, Issue 1, pp 19–32 | Cite as

Purification and characterization of an exoinulinase from Aspergillus fumigatus

  • Prabhjot Kaur Gill
  • Rajesh Kumari Manhas
  • Jatinder Singh
  • Prabhjeet SinghEmail author
Original Articles


An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K m (0.25 mM) and higher V max (333.3 IU/mg) values for inulin.

Index Entries

Inulin exoinulinase fructose Aspergillus fumigatus concanavalin A 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Chen, W. C. and Liu, C. H. (1996), Enzyme Microb. Technol. 1296, 153–160.CrossRefGoogle Scholar
  2. 2.
    Elmer, G. W. (1986), JAMA 275, 870–876.CrossRefGoogle Scholar
  3. 3.
    Hatcher, G. E. and Lambrecht, R. S. (1993), J. Dairy Sci. 76, 2485–2492.PubMedCrossRefGoogle Scholar
  4. 4.
    Vandamme, E. J. and Derycke, D. G. (1983), Adv. Appl. Microbiol. 29, 139–176.PubMedCrossRefGoogle Scholar
  5. 5.
    Kaur, A., Sharma, D., Harchand, R. K., Singh, P., Bhullar, S. S., and Kaur, A. (1999), Indian J. Microbiol. 39, 99–103.Google Scholar
  6. 6.
    Sharma, D., Kaur, A., Harchand, R. K., Singh, P., Bhullar, S. S., and Kaur, A. (1998), Indian J. Microbiol. 38, 235, 236.Google Scholar
  7. 7.
    Bradford, M. M. (1976), Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  8. 8.
    Nelson, N. (1944), J. Biol. Chem. 153, 375–380.Google Scholar
  9. 9.
    Singh, J., Kamboj, S. S., Sandhu, R. S., Shangary, S., and Kamboj, K. K. (1993), Phytochemistry 33, 979–983.CrossRefGoogle Scholar
  10. 10.
    Laemmli, U. (1970), Nature 227, 680–685.PubMedCrossRefADSGoogle Scholar
  11. 11.
    Gabriel, O. and Wang, S. F. (1969), Anal. Biochem. 27, 545–554.PubMedCrossRefGoogle Scholar
  12. 12.
    Celis, J. E., Lauridsen, J. B., and Basse, B., eds. (1994), in Cell Biology: A Laboratory Handbook, 2nd ed., Academic, San Diego, pp. 305–313.Google Scholar
  13. 13.
    Trevelyan, W. E., Procter D. P., and Harrison, J. S. (1950), Nature 166, 444, 445.PubMedCrossRefADSGoogle Scholar
  14. 14.
    Ettalibi, M. and Baratti, J. C. (1987), Agric. Biol. Chem. 54, 61–68.Google Scholar
  15. 15.
    Kochhar, A., Gupta, A. K., and Kaur, N. (1999), J. Sci. Food Agric. 79, 549–554.CrossRefGoogle Scholar
  16. 16.
    Kochhar, A., Kaur, N., and Gupta, A. K. (1997), J. Sci. Ind. Res. 57, 184–187.Google Scholar
  17. 17.
    Arand, M., Golubev, A. M., Neto, J. R. B., et al. (2002), Biochem. J. 362, 131–135.PubMedCrossRefGoogle Scholar
  18. 18.
    Nakamura, T., Kurokawa, T., Nakatsu, S., and Ueda, S. (1978), Nippon. Nogeikagaku. Kaishi. 52, 581–587.Google Scholar
  19. 19.
    Uhm, T. A. and Byun, S. M. (1987), Biotechnol. Lett. 9, 287–290.CrossRefGoogle Scholar
  20. 20.
    Beluche, I., Guiraud, J. P., and Galzy, P. (1980), Folio Microbiol. 25, 32–39.Google Scholar
  21. 21.
    Guirad, J. P., Viard-Gaudin, C., and Galzy, P. (1980), Agric. Biol. Chem. 44, 245–252.Google Scholar
  22. 22.
    Derycke, D. G. and Vandamme, E. J. (1984), J. Chem. Tech. Biotechnol. 34, 45–51.Google Scholar
  23. 23.
    Pessoni, R. A. B., Figueiredo, R., and Braga, M. R. (1999), J. Appl. Microbiol. 87, 141–147.PubMedCrossRefGoogle Scholar
  24. 24.
    Efstathiou, I., Reysset, G., and Truffaut, N. (1986), Appl. Microbiol. Biotechnol. 25, 143–149.Google Scholar
  25. 25.
    Kaur, N., Kaur, M., Gupta, A. K., and Singh, R. (1992), J. Chem. Tech. Biotechnol. 53, 279–284.Google Scholar
  26. 26.
    Hazaa, M. M. (1999), J. Union Arab. Biol. 8, 467–482.Google Scholar
  27. 27.
    Nakamura, T., Ogata, Y., Shitara, A., Nakamura, A., and Ohta, K. (1995), J. Ferment. Bioeng. 80, 164–169.CrossRefGoogle Scholar
  28. 28.
    Nakamura, T., Shitara, A., Matsuda, S., Matsuo, T., Suiko, M., and Ohta, K. (1997), J. Ferment. Bioeng. 84, 313–318.CrossRefGoogle Scholar
  29. 29.
    Nahm, B. H. and Byun, M. (1977), Korean Biochem. J. 10, 95–108.Google Scholar
  30. 30.
    Mukherjee, K. and Sengupta, S. (1987), Can. J. Microbiol. 33, 520–524.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Prabhjot Kaur Gill
    • 1
  • Rajesh Kumari Manhas
    • 2
  • Jatinder Singh
    • 3
  • Prabhjeet Singh
    • 1
    Email author
  1. 1.Department of BiotechnologyGuru Nanak Dev UniversityPunjabIndia
  2. 2.Department of MicrobiologyGuru Nanak Dev UniversityPunjabIndia
  3. 3.Department BiochemistryGuru Nanak Dev UniversityPunjabIndia

Personalised recommendations