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

Abstract

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±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/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 P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06

“…As shown in Fig. 6, exoinulinase showed maximum activity at pH 4.5, but endoinulinase exhibited the highest activity at pH 5.0, which was in agreement with the general range of many microbial sources reported so far: A. niger (pH 4.4) (Derycke & Vandamme, 1984), A. Awamori (pH 4.5) (Arand et al, 2002), Penicillium janczewskii (pH 4.8-5.0) (Pessoni, Figueiredo, & Braga, 1999), Penicillium sp. TN-88 (pH 5.2) (Nakamura et al, 1997).…”

Crystal Structure of Exo-inulinase from Aspergillus awamori: The Enzyme Fold and Structural Determinants of Substrate Recognition

“…Crystallization of the enzyme in the primitive orthorhombic P2 1 2 1 2 1 space group has been reported. 14 The new crystal form belongs to the monoclinic P2 1 space group. The crystallographic structure of the enzyme was solved by the SIRAS method, using a quick cryo-soaking technique, 19-21 at 1.55 Å resolution in the monoclinic form and was refined to the final R work and R free of 17.0% and 19.8%, respectively.…”

“…Successful biotechnological applications of inulinases have brought significant attention to these enzymes. 8 Inulinases expressed by Penicillium, [9][10][11] Kluyveromyces, 12 and Aspergillus 13,14 are the most intensively studied fungal enzymes of this class. Exo-inulinases (EC 3.2.1.80, also known as fructan b-fructosidase or exo-b-D-fructosidase) hydrolyze terminal, nonreducing 2,1-linked and 2,6-linked b-Dfructofuranose residues in fructans, with concomitant release of b-D-fructose.…”

Purification and characterization of heat-stable exo-inulinase from Streptomyces sp.

“…Fructose was found to be the only product of inulin hydrolysis, which suggests that purified inulinase is an exo-inulinase. In this respect, it resembles the inulinases of Bacillus polymyxa (Kwon et al, 2003), A. awamori (Arand et al, 2002) and Geobacillus stearothermophilus (Tsujimoto et al, 2003).…”