Purification and Characterization of Aspergillus ficuum Endoinulinase.

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  • UHM Tai-Boong
    Faculty of Biological Sciences and Institute for Molecular Biology and Genetics, Chonbuk National University
  • CHUNG Mi Sun
    Department of Food Science and Technology, Chonbuk National University
  • LEE Sun Hee
    Department of Food Science and Technology, Chonbuk National University
  • GOURRONC Françoise
    Laboratoire de Biochimie, Université de Reims, UFR Sciences
  • HOUSEN Isabelle
    Laboratoire de Génétique Moléculaire, Facultés Universitaires
  • KIM Jong Hwa
    Department of Food Science and Engineering, Woo Suk University
  • BEEUMEN Josef Van
    Laboratorium voor Eiwitbiochimie en Eiwitengineering, University of Gent
  • HAYE Bernard
    Laboratoire de Biochimie, Université de Reims, UFR Sciences
  • VANDENHAUTE Jean
    Laboratoire de Génétique Moléculaire, Facultés Universitaires

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  • Purification and Characterization of<i>Aspergillus ficuum</i>Endoinulinase

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Abstract

  Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000±500 and 66,000±1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800±1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1±1.0 mM and 773±60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.<br>

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