|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
This project concerns "apidaecin" of honey bee origin, a model of antimicrobial peptides.It is composed of l8 amino acid residues and has a potential of being applied to the protection agianst pathogenic infection to plants and animals, and also applied to the food preservative. The aim of this project is to develop experimental evolution systems for the above-mentioned purposes.The research results obtained were as follows. First, we succeeded in obtaining many apidaecin mutants with lower activities by combining localized random mutagenesis for the apidaecin gene and originally developed in vivo assay-screening systems. By using these mutants, functional mapping became possible.indicating, e.g.the significant roles of the C-terminal region and Pro・Arg residues of apidaecin molecule. The relationship between the in vivo and in vitro assay systems and usefulness of the ampicillin screening for obtaining higher active apidaecin mutants were also verified (the first year). Next, in the second year.we carried out some preparative studies, e.g., trials to improve the microbial production of apidaecin.to obtain higher active apidaecin mutants, and also to clarify the mechanism of antimicrobial action. In the third year.based on the results obtained in the past two years, we succeeded in obtaining the higher active apidaecin mutants by chemically synthesizing apidaecin genes which brought about randomized amino acid residues not so essential for the antimicrobial action. Further, we tried to apply all the same systems as have been developed so far to an antimicrobial peptide other than apidaecin : thanatin of schield bug origin, which is effective against not only gramnegative and -positive bacteria but also fungi. As the result, we could obtain many thanatin mutants, and thus verify that our experimental evolution systems can be universally useful for improving any antimicrobial peptide.