Molecular Cloning, Functional Expression, and Mutagenesis of cDNA Encoding Class I Chitinase from Rye (Secale cereale) Seeds

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著者

    • ASO Yoichi
    • Laboratory of Protein Chemistry and Engineering, Graduate School of Bioresource and Environmental Science, Kyushu University
    • ISHIGURO Masatsune
    • Laboratory of Protein Chemistry and Engineering, Graduate School of Bioresource and Environmental Science, Kyushu University

抄録

A cDNA encoding rye seed chitinase-a (RSC-a) was cloned by rapid amplification of cDNA ends and PCR procedures. It consists of 1,191 nucleotides and encodes an open reading frame of 321 amino acid residues. Recombinant RSC-a (rRSC-a) was produced in the oxidative cytoplasm of <I>Escherichia coli</I> Origami(DE3) in a soluble form by inducing bacteria at a low temperature (20°C). Purified rRSC-a showed properties similar to the original enzyme from rye seeds in terms of chitinase activity toward a soluble substrate, glycolchitin, and an insoluble substrate, chitin beads, in chitin-binding ability to chitin, and in antifungal activity against <I>Trichoderma</I> sp. <I>in vitro.</I> rRSC-a mutants were subsequently produced and purified by the same procedures as those for rRSC-a. Mutation of Trp23 to Ala decreased the chitinase activity toward both substrates and impaired the chitin-binding ability. Furthermore, the antifungal activity of this mutant was weakened with increasing of the NaCl concentration in the culture medium. Complete abolishment of both activities was observed upon the mutation of Glu126 to Gln. The roles of these residues in both activities are discussed.

収録刊行物

  • Bioscience, biotechnology, and biochemistry

    Bioscience, biotechnology, and biochemistry 68(2), 324-332, 2004-02-23

    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)
    10013141811
  • 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
    6868370
  • 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  CJP引用  NDL  J-STAGE 
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