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 +/- 1 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 in the hydrolysis of inulin were 0.003 +/- 0.0001 mM and 175 +/- 5 mumol (.) min(-1) (.) mg(-1). The same parameters in the hydrolysis of levan were 2.08 +/- 0.04 mg/ml and 1.2 +/- 0.02 mumol/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 Aspergillis 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 Angstrom (1Angstrom = 0.1 nm), b = 82.041 Angstrom and c = 136.075 Angstrom. Crystals diffracted beyond 1.54 Angstrom, and useful X-ray data were collected to a resolution of 1.73 Angstrom.