title: Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis.
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 beta2,1- as well as beta2,6-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.
authors.affiliation: Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina, Orlova Rosha, St. Petersburg 188350, Russia.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
authors.forename: Michael
authors.initials: M
authors.lastname: Arand
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eneyskaya
authors.forename: Dina R
authors.initials: DR
authors.lastname: Ivanen
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Shabalin
authors.forename: Sergei M
authors.initials: SM
authors.lastname: Shishlyannikov
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Saveliev
authors.forename: Olga S
authors.initials: OS
authors.lastname: Korneeva
authors.forename: Kirill N
authors.initials: KN
authors.lastname: Neustroev
mesh_terms: D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism*; D001665:Binding Sites; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007444:Inulin / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013997:Time Factors
chemical_list: D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase