Molecular cloning and sequence analysis of an endoinulinase gene from Penicillium sp. strain TN-88.
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 beta-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase.
Akimoto
H
H
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, Japan.
Kiyota
N
N
Kushima
T
T
Nakamura
T
T
Ohta
K
K
D020413:3' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis; D005656:Fungal Proteins; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D007444:Inulin / Q000378:metabolism; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology / Q000235:genetics*; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D043324:beta-Fructofuranosidase
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D012333:RNA, Messenger; D007444:Inulin; D006026:Glycoside Hydrolases; C421180:InuC protein, Penicillium; D043324:beta-Fructofuranosidase