Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Biochemistry & Molecular Biology Regular Papers
Rice Bifunctional α-Amylase/Subtilisin Inhibitor: Cloning and Characterization of the Recombinant Inhibitor Expressed in Escherichia coli
Teruyuki YAMASAKIMasaki DEGUCHIToshiko FUJIMOTOTakehiro MASUMURATomohide UNOKengo KANAMARUHiroshi YAMAGATA
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2006 Volume 70 Issue 5 Pages 1200-1209

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Abstract

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.

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© 2006 by Japan Society for Bioscience, Biotechnology, and Agrochemistry
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