Cloning of the Ruminococcus albus cel5D and cel9A Genes Encoding Dockerin Module-containing Endoglucanases and Expression of cel5D in Escherichia coli

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An <I>Eco</I>RI chromosomal DNA fragment of <I>Ruminococcus albus</I> F-40 that conferred endoglucanase activity on <I>Escherichia coli</I> was cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the <I>Eco</I>RI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated <I>cel5D</I> and <I>cel9A</I> respectively. The <I>cel5D</I> gene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The <I>cel9A</I> gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant <I>E. coli</I>. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a <I>K</I>m of 3.9 mg/ml and a <I>V</I>max of 37.2 μmol/min/mg for carboxymethycellulose.

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  • Bioscience, biotechnology, and biochemistry

    Bioscience, biotechnology, and biochemistry 68(7), 1557-1564, 2004-07-23

    公益社団法人 日本農芸化学会

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各種コード

  • NII論文ID(NAID)
    10013520189
  • NII書誌ID(NCID)
    AA10824164
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09168451
  • NDL 記事登録ID
    7031304
  • NDL 雑誌分類
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL 請求記号
    Z53-G223
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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