Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae.
OBJECTIVE
Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties.


RESULTS
The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co(2+)-containing medium. The enzyme was extracted from the 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 reactivity towards peptide substrate rather than synthetic substrates.


CONCLUSIONS
Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co(2+)-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis.


CONCLUSIONS
Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.
Kusumoto
K-I
KI
National Food Research Institute, Tsukuba, Ibaraki, Japan. kusumoto@affrc.go.jp
Matsushita-Morita
M
M
Furukawa
I
I
Suzuki
S
S
Yamagata
Y
Y
Koide
Y
Y
Ishida
H
H
Takeuchi
M
M
Kashiwagi
Y
Y
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D043384:Glutamyl Aminopeptidase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D003470:Culture Media; D011994:Recombinant Proteins; D003035:Cobalt; D043384:Glutamyl Aminopeptidase; C018021:cobaltous chloride