Efficient production and partial characterization of one of metalloproteases, aspartyl aminopeptidase from Aspergillus oryzae

Ken-Ichi Kusumoto1, Mayumi Matsushita1, Ikuyo Furukawa1, Satoshi Suzuki1, Youhei Yamataga2, Yoshinao Koide3, Hiroki Ishida4, Michio Takeuchi5, Yutaka Kashiwagi1

Author address: 

1National Food Research Institute, Tsukuba, Ibaraki, Japan, 2Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan, 3Gifu R & D Center, Amano Enzyme Inc., Kagam


Aspartyl aminopeptidase from Aspergillus oryzae has high substrate specificity, degrading only amino-terminal acidic amino acids from peptides, and may be suitable for processing physiological peptides. However, little is known about the biochemical properties of this enzyme and its efficient production in Aspergillus oryzae. The gene encoding aspartyl aminopeptidase was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a cobalt-containing medium. The enzyme was extracted from mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high affinity to peptide substrate rather than synthetic substrates. Thus, recombinant A. oryzae aspartyl aminopeptidase was purified to homogeneity with an increased specific activity, when cultivated in a cobalt ion-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help increase specific activity of other metalloproteases and their functional analysis

abstract No: 


Full conference title: 

    • ECFG 9th (2008)