ツクツクボウシタケ由来プロテアーゼの精製とその性質 Purification and characterization of a protease from Isaria cicadae

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Author(s)

    • 竹川 千裕 TAKEKAWA Chihiro
    • 山梨大学大学院医学工学総合研究部 Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
    • 中村 和夫 NAKAMURA Kazuo
    • 山梨大学大学院医学工学総合研究部 Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
    • 本間 裕人 [他] HOMMA Hiroto
    • 東京農業大学短期大学部醸造学科 Department of Brewing and Fermentation, Junior College of Tokyo University of Agriculture
    • 大山 拓次 OYAMA Takuji
    • 山梨大学大学院医学工学総合研究部 Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi

Abstract

ツクツクボウシタケのフスマ液体培養から抽出した菌体外プロテアーゼを精製し,その性質を調べた.寒天培地上で分生子形成菌糸体と未分生子形成菌糸体の二形態をとることがわかった.分生子形成菌糸体は高い酵素活性を示した.CM-Sepharoseカラムクロマトグラフィーによって,収率33%,13倍に精製された.酵素標品は電気泳動的に単一のバンドが得られた.分子量はSDS-PAGEによって30.6kDa,ゲル濾過クロマトグラフィーによって31kDaであったので,本酵素はモノマーであることが示唆された.N-末端アミノ酸配列はAFTTQPGAVWであった.最適温度と最適pHはそれぞれ55℃と9.0であった.pH4-7の範囲で安定であった.カゼインに対するKm値は0.48mg/mLであった.活性はPMSFによって強く阻害された.これらの結果から本酵素はセリンプロテアーゼであることが示された.

An extracellular protease in Isaria cicadae extracted from a liquid culture with wheat bran was purified and characterized. This mushroom showed two morphologies on agar plates: mycelia with and without conidia. High enzyme activity was detected in the culture filtrate extract of the mycelia with conidia. The enzyme was purified 13-fold with a yield of 33% using CM-Sepharose column chromatography. Electrophoretic analysis of the purified enzyme showed a single band. The molecular mass of the enzyme was 30.6 kDa by SDS-PAGE and 31 kDa by gel filtration. The N-terminal amino acid sequence of the protease was AFTTQPGAVW. The optimum temperature and pH were 55℃ and 9.0, respectively. The enzyme was stable in the pH range 4 to 7. The Km value for the hydrolysis of casein was 0.48mg/mL. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride. From the results, it is suggested that the enzyme is a serine protease.

Journal

  • Mushroom Science and Biotechnology

    Mushroom Science and Biotechnology 22(2), 74-78, 2014

    Japanese Society of Mushroom Science and Biotechnology

Codes

  • NII Article ID (NAID)
    110009863113
  • NII NACSIS-CAT ID (NCID)
    AA12415767
  • Text Lang
    ENG
  • ISSN
    1348-7388
  • NDL Article ID
    025776177
  • NDL Call No.
    Z18-B266
  • Data Source
    NDL  NII-ELS  J-STAGE 
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