Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

• Published in: Mycoscience Vol. 53; no. 5; pp. 354 - 364
• Main Authors: Shirasaka, Norifumi, Naitou, Masao, Okamura, Kazuki, Fukuta, Yasuhisa, Terashita, Takao, Kusuda, Mizuho
• Format: Journal Article
• Language: English
• Published: The Mycological Society of Japan 2012
• Subjects: Edible fungi; Fibrin; Koji; Serine protease
• ISSN: 1340-3540; 1618-2545
• DOI: 10.47371/S10267-011-0179-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.