Assuming that Asp225 is the substrate specificity determinant of Achromobacter protease I (API) which is lysine-specific serine protease, the 225th residue was substituted for other amino acids with a hope that the substrate specificity of a mutant API is altered. Furthermore, to maturate proform of mutant API autocatalytically, Lys(-1) was also replaced by Met, Asp, or Glu. However, all the mutants were not expressed, or accumulated as inactive precursor proteins. This result implicats that Asp225 plays a critical rol in restricted substrate specificity as a lysylendopeptidase but the substrate specificity of API is not determined only by the nature of residue 225.