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An aminopeptidase with original specificity was purified 3800-fold to homogeneity from a cellular extract of Staphylococcus chromogenes. The enzyme was specific for acidic amino acids (Asp and Glu) at the N-terminus of peptides and thus can be classified as an aminopeptidase A. However, its specificity was not restricted to acidic amino acids: α-hydroxy acids such as L-malic and L-lactic acids were also accepted in position P1. The enzyme had a broad specificity for the residue at position P' 1, accepting all types of amino acids, including Pro, in this position. The optimal conditions for the hydrolysis of Asp-Phe-NH2 were pH 9.5 and 60°C. The enzyme was inhibited by chelating agents and serine-protease inhibitors. The activity lost by treatment with chelating agents could be restored by Mn2+ or Zn2+ which also stimulated the native enzyme. This suggests that it is a metalloprotease with a serine residue essential for the activity. The native enzyme had an apparent molecular mass of 430 kDa on gradient-gel electrophoresis and subunits of 43 kDa as determined by SDS/PAGE. The enzyme catalyzed the synthesis of peptide and amino acid derivatives such as Asp-Phe-OMe (Aspartame) and malyl-Tyr-OEt from L-Asp and L-malic acid as acyl donors and L-Phe-OMe and L- Tyr-OEt as nucleophiles, respectively. The use of the enzyme as a reagent in protease-catalyzed peptide synthesis, N-terminal protection and subsequent deprotection, is described. [ABSTRACT FROM AUTHOR]