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Biochemical characterization ofAspergillus awamoriexoinulinase: substrate binding characteristics and regioselectivity of hydrolysis

Authors
Journal
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
1570-9639
Publisher
Elsevier
Publication Date
Volume
1650
Identifiers
DOI: 10.1016/s1570-9639(03)00187-0
Keywords
  • Exoinulinase
  • Fructooligosaccharide
  • Fructosyl-Binding Site
  • Multiple Attack
Disciplines
  • Biology

Abstract

Abstract 1H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity. Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme hydrolyzed both β2,1- as well as β2,6-fructosyl linkages in fructooligosaccharides. From the k cat/ K m ratios obtained with inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase.

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