Purification and characterization of intracellular serine protease produced in mycelia during fruit-body growth stage of Hypsizygus marmoreus

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  • FUKUTA Yasuhisa
    Laboratory of Food Microbiological Science and Biotechnology, Faculty of Agriculture, Kin-Ki University
  • ITO Katsumi
    Laboratory of Food Microbiological Science and Biotechnology, Faculty of Agriculture, Kin-Ki University
  • SHIRASAKA Norifumi
    Laboratory of Food Microbiological Science and Biotechnology, Faculty of Agriculture, Kin-Ki University
  • KUSUDA Mizuho
    Laboratory of Biocycle Engineering, Graduate School of Life and Environmental Sciences, Osaka Prefectural University
  • MIHARA Satoshi
    Japan Agricultural Cooperatives of Nakano city
  • YAMANAKA Katsuji
    Kyoto Mycological Institute
  • TERASHITA Takao
    Laboratory of Food Microbiological Science and Biotechnology, Faculty of Agriculture, Kin-Ki University

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Other Title
  • ブナシメジ子実体形成時における菌糸体内セリンプロテアーゼの精製と酵素化学的諸性質の解明

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The purification and characterization of a neutral protease (PE-4) produced in mycelia during fruit-body formation in Hypsizygus marmoreus was carried out in the present study. The enzyme was purified to homogeneity using ammonium fractionation, ion exchange chromatography on DEAE-Toyopearl, hydrophobic exchange chromatography on Butyl-Toyopearl, and gel chromatography on Superdex 200. Protease activity was enhanced by Co^<2+>, in particular, and was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), aprotinin and Pefabloc SC, which are known serine protease inhibitors. Phosphoramidon did not inhibit the protease. The N-terminal amino acid sequence was identical with the serine protease from the fruit-body of H. marmoreus, hmsp. The same serine protease was present in both vegetative mycelia and the fruit-body in the growth stage. Protease activity activated in mycelia at fruit-body formation was inhibited by PMSF, EDTA, and phosphoramidon but not completely. These results also suggest that several neutral proteases besides PE-4 were present during kinkaki, which is removal of both the spawn and the uppermost layer of the medium, in the mature fruit-body in the mycelia.

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