BBB : Vol. 64 (2000) , No. 11 pp.2328-2335

Molecular Cloning and Sequence Analysis of an Endoinulinase Gene from Penicillium sp. Strain TN-88

Hidetoshi AKIMOTO¹⁾, Naoyuki KIYOTA¹⁾, Takayuki KUSHIMA¹⁾, Toyohiko NAKAMURA¹⁾ and Kazuyoshi OHTA¹⁾

1) Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University

(Received April 17, 2000)
(Accepted July 19, 2000)

A genomic DNA segment and cDNAs encoding an extracellular endoinulinase of Penicillium sp. strain TN-88 were cloned and sequenced. Southern blot analysis indicated that the endoinulinase gene (inuC) was present as a single copy in the genome. An open reading frame, consisting of 1,545 bp, was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. Three distinct transcriptional start points were observed at positions -242 (A), -215 (A), and -75 (C) from the start codon. The 5′-noncoding region had a putative TATA box at position -120 (TATATATA) and two contiguous CAAT sequences at -159 to -151. The deduced amino acid sequence showed 72 and 85% identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct cluster from other members of the β-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase.

Key words:

cDNA; endoinulinase; inulin; Penicillium sp.; phylogenetic analysis