Purification and Some Properties of an Alkaline Protease Inhibitor-Inactivating-Enzyme from Aspergillus oryzae W-1

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  • Ishihara Masanobu
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus
  • Morine Nobuya
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus
  • Katayama Hisao
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus
  • Gima Tomoyori
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus
  • Taira Toki
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus
  • Tawata Shinkichi
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus
  • Kobamoto Naotada
    Department of Bioscience and biotechnology, Faculty of Agriculture, University of the Ryukyus

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  • Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ　Ｗ‐１のアルカリプロテアーゼインヒビター失活化酵素の精製と性質
  • Aspergillus oryzae W 1 ノ アルカリプロテアーゼインヒビター シッカツカ コウソ ノ セイセイ ト セイシツ

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Abstract

An alkaline protease inhibitor-inactivating enzyme (AIE) of Aspergillus oryzae W-1 was purified to homogeneity by the combination of various column chromatographies. The molecular mass of the enzyme was estimated to be 42 kDa by SDS-PAGE; the isoelectric point was 4.7. AIE was optimally active at around 55°C and pH6. The enzyme was stable up to 40°C and in the pH range of 6-10. AIE was inactivated by HgCl2 or p-chloromercuribenzoate (p-CMB) but not by trypsin inhibitor, leupeptin, and phenylmethylsulfonylfluoride (PMSF). It was also inactivated by chelating agents, like ethylenediaminetetraacetate (EDTA) and o-phenanthroline. The enzyme treated with HgCl2, however, was reactivated by the addition of 2-mercaptoethanol (2-ME). On the other hand, EDTA-inactivated AIE was reactivated in the presence of 2-ME and CaCl2. When the protease-inhibitor complex was incubated with AIE at 37°C and pH5, the activation of AP was detected. From these results, it was supposed that AIE is responsible for the activation of alkaline protease (AP) in the cells of mold.

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