Rice Bifunctional α-Amylase/Subtilisin Inhibitor : Cloning and Characterization of the Recombinant Inhibitor Expressed in Escherichia coli

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概要

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• The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional α-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to α-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial α-amylases, but not toward animal or insect α-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.

• 社団法人 日本農芸化学会の論文
• 2006-05-23

著者

• UNO Tomohide

  Laboratory of Biochemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe Univ

• Kanamaru Kengo

  Laboratory of Molecular Genetics, Institute of Molecular and Cellular Biosciences, The University of

• MASUMURA Takehiro

  Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefect

• Yamagata Hiroshi

  Laboratory Of Biological Chemistry Faculty Of Agriculture Kobe University

• Yamagata Hiroshi

  Laboratory Of Biochemistry Faculty Of Agriculture Kobe University

• Uno Tomohide

  Laboratory Of Biochemistry Department Of Biofunctional Chemistry Faculty Of Agriculture Kobe Univers

• Uno Tomohide

  Laboratory Of Biochemistry Faculty Of Agriculture Kobe University

• Kanamaru Kengo

  Laboratory Of Biochemistry Faculty Of Agriculture Kobe University

• Kanamaru Kengo

  Lab. Of Biochemistry Graduate School Of Agricultural Sci. Kobe Univ.

• YAMASAKI Teruyuki

  Laboratory of Biochemistry, Faculty of Agriculture, Kobe University

• DEGUCHI Masaki

  Laboratory of Biochemistry, Faculty of Agriculture, Kobe University

• FUJIMOTO Toshiko

  Laboratory of Biochemistry of Biomacromolecules, Graduate School of Science and Technology, Kobe Uni

• Masumura Takehiro

  Laboratory Of Genetic Engineering Graduate School Of Agriculture Kyoto Prefectural University

• Deguchi Masaki

  Laboratory Of Biochemistry Faculty Of Agriculture Kobe University

• Yamagata Hiroshi

  Laboratory Of Biochemistry Graduate School Of Agricultural Science Kobe University

• Fujimoto Toshiko

  Laboratory Of Biochemistry Of Biomacromolecules Graduate School Of Science And Technology Kobe Unive

• Yamasaki Teruyuki

  Laboratory Of Biochemistry Faculty Of Agriculture Kobe University

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