Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis.

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.

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori.

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69 +/- kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the beta-(2-->1)-fructan (inulin) and beta-(2-->6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants K(m) and V(max) in the hydrolysis of inulin were 0.003 +/- 0.0001 mM and 175 +/- 5 micromol.min(-1).mg(-1). The same parameters in the hydrolysis of levan were 2.08 +/- 0.04 mg/ml and 1.2 +/- 0.02 micromol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P2(1)2(1)2(1) with cell parameters a=64.726 A (1A=0.1 nm), b=82.041 A and c=136.075 A. Crystals diffracted beyond 1.54 A, and useful X-ray data were collected to a resolution of 1.73 A.