Bacillus subtilis M2-4 由来タンパク分解酵素の精製とその性質 Purification and Characterization of a Proteolytic Enzyme from Bacillus subtilis M2-4

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Abstract

パンに代表される伝統的な自然発酵食品は,古くから世界中で食され,自然発酵微生物の生産するタンパク質分解酵素はパン作りに利用されてきた.今回は,小麦粉発酵食品よりタンパク分解酵素を産生するグラム陽性桿菌B.subtilis M2-4を分解した.分離菌株は, 16S rRNAによる遺伝子解析の結果,B.subtilisと99%一致した.B.subtilis M2-4の産生するタンパク分解粗酵素は,カラムクロマトグラフィーを用い,単一バンドまで精製した.ゲルろ過により精製酵素の分子量は,33kDaと推定された.至適温度は55℃で,至適pHは11.0であり,その酵素安定性は,50℃まで,ならびにpH5.0-11.0の範囲で安定であった.N-末端アミノ酸配列はAQSVPYGISQIKAPAであり,サブチリシンと同じであった.しかし,小麦粉発酵食品が分離源であることは初めてであった.酸性カゼインに対する分解フラッグメントのC末端は,特に親水性アミノ酸アルギニン,グルタミン,疎水性アミノ酸バリン,イソロイシン等のアミノ酸であった.

The proteolytic enzymes produced by naturally occurring microorganisms during the bread-making process were investigated. We isolated one Gram-positive and aerobic endospore-forming rod bacterium designated B. <I>subtilis</I> M2-4 producing a proteolytic enzyme from traditional and naturally fermented wheat flour. An analysis of the 16S rRNA sequence of the isolated strain revealed it to be 99% identical to <I>Bacillus subtilis</I>. We purified the proteolytic enzyme to electrophoretic homogeneity from the culture supernatant by column chromatography. The calculated molecular mass of the purified enzyme determined by gel filtration was 33 kDa. The proteolytic enzyme showed optimal activity at 55°Cand pH 11.0, and was stable below 50°C and in the pH range of 5.0-11.0. The <I>N</I>-terminal amino acid sequence of the purified protease from <I>B. subtilis</I> M2-4 was AQSVPYGISQIKAPA, the same as other subtilisins. However, this is the first report of its isolation from traditional fermented wheat flour. The purified protease digested acid casein into fragments with hydrophilic and hydrophobic amino acids at the C-terminal, in particular Arg, Glu, Val and Ile.

Journal

  • Journal of Home Economics of Japan

    Journal of Home Economics of Japan 59(8), 565-573, 2008-08-01

    The Japan Society of Home Economics

References:  28

Cited by:  1

Codes

  • NII Article ID (NAID)
    110006862307
  • NII NACSIS-CAT ID (NCID)
    AN10040097
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    09135227
  • NDL Article ID
    9601450
  • NDL Source Classification
    ZE16(社会・労働--家事・家政--学術誌)
  • NDL Call No.
    Z6-113
  • Data Source
    CJP  CJPref  NDL  NII-ELS  IR  J-STAGE  NDL-Digital 
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