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

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Mycoscience

Abstract

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.

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Authors and Affiliations

  1. Laboratory of Food Microbiological Science and Biotechnology, Graduate School of Agriculture, Kin-ki University, 3327-204 Nakamachi, Nara, 631-8505, Japan

    Norifumi Shirasaka, Masao Naitou, Kazuki Okamura, Yasuhisa Fukuta & Takao Terashita

  2. Laboratory of Biocycle Engineering, Graduate School of Life and Environmental Sciences, Osaka Prefectural University, 1-1 Gakuencho, Naka-ku, Sakai, Osaka, 599-8531, Japan

    Mizuho Kusuda

Authors
  1. Norifumi Shirasaka
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  2. Masao Naitou
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  3. Kazuki Okamura
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  4. Mizuho Kusuda
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  5. Yasuhisa Fukuta
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  6. Takao Terashita
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Correspondence to Norifumi Shirasaka.

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Shirasaka, N., Naitou, M., Okamura, K. et al. Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3. Mycoscience 53, 354–364 (2012). https://doi.org/10.1007/s10267-011-0179-3

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  • DOI: https://doi.org/10.1007/s10267-011-0179-3

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