Molecular cloning of ocpO encoding carboxypeptidase O of Aspergillus oryzae IAM2640.
Carboxypeptidase O from Aspergillus oryzae IAM2640 is a serine-type carboxypeptidase. In this study, we cloned and sequenced cDNA and genomic DNA carrying ocpO encoding carboxypeptidase O. The results showed that the length of ocpO was 1,816 bp, and the open reading frame encoded a putative preproenzyme composed of 472 amino acid residues of the mature carboxypeptidase O and an additional N-terminal sequence of 50 amino acid residues. A BLASTN search revealed that a gene, AO090020000351, in A. oryzae RIB40, which is strain used in genome-wide sequencing, is a homolog of ocpO. The difference between AO090020000351 and ocpO was only one nucleotide. The difference caused substitution of Ala for Pro at the 277th position of the enzyme; therefore the protein encoded by AO090020000351 was overproduced and purified. The purified protein showed enzymatic properties similar to carboxypeptidase O, indicating that carboxypeptidase O and protease encoded by AO090020000351 are same enzyme.
Morita
Hiroto
H
Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
Kuriyama
Ken-Ichi
K
Akiyama
Noritaka
N
Okamoto
Ayako
A
Yamagata
Youhei
Y
Kusumoto
Ken-Ichi
K
Koide
Yoshinao
Y
Ishida
Hiroki
H
Takeuchi
Michio
M
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D030541:Databases, Genetic; D004795:Enzyme Stability; D016681:Genome, Fungal / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D004271:DNA, Fungal; D011480:Protease Inhibitors; D002268:Carboxypeptidases