Purification and properties of an extracellular exoinulinase from Penicillium sp. strain TN-88 and sequence analysis of the encoding gene.
An exoinulinase, P-I, was purified from the culture filtrate of Penicillium sp. strain TN-88 grown on inulin. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis with an apparent Mr of 81 kDa. The purified enzyme had extremely high specific activity, 743 U/mg, toward inulin. Inulinase activity was optimal at pH 4.0 and 55 degrees C. A genomic DNA and cDNAs encoding this protein were cloned and sequenced. The exoinulinase gene (inuD) was present as a single copy in the genome. An open reading frame of 2,106 bp was interrupted by a single intron of 56 bp, and encoded a 25-amino acid signal peptide and a 677-amino acid mature protein. The mature protein contained two Cys residues and eight potential N-linked glycosylation sites. The 5'-noncoding region had a putative CAAT box at position -239. Four distinct transcription start points were observed at positions -98 (A), -91 (A), -80 (A), and -76 (A) from the start codon. The exoinulinase gene inuD was located 860-bp upstream of the previously reported endoinulinase gene inuC in the opposite direction of transcription.
Moriyama
Satoshi
S
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, Japan.
Akimoto
Hidetoshi
H
Suetsugu
Norio
N
Kawasaki
Soushi
S
Nakamura
Toyohiko
T
Ohta
Kazuyoshi
K
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005800:Genes, Fungal / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase