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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

Abstract

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 

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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

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