Substrate Specificities of Aminopeptidases: A Specific Method for Microbial Differentiation

The substrate specificities of aminopeptidases in various species of bacteria, parasites, and fungi have been shown to be radically different. These differences have been used to distinguish closely related species and even strains of the same species. The aminopeptidase (arylamidase) procedure is based upon the enzymatic liberation of fluorescent β-naphthylamine (βNA) from a nonfluorescent L-amino-acid-β-naphthylamide. This allows quantitative and qualitative measurements as fluorescence is linear. Bacteria, fungi, and parasites are differentiated on their ability to enzymatically hydrolyze a series of L-amino-acid-βNAs producing a specific, characteristic profile. Differentiation by substrate specificity of the aminopeptidases complements and sometimes supercedes morphological and cultural identification techniques. It does not suffer from data interpretational difficulties that are sometimes caused by interfering chemical constituents in electrophoretic, chromatographic, serological, or phosphorescent techniques. The chapter presents preparation of cell-free aminopeptidases from Enterobacteriaceae and profile determination with cell-free aminopeptidases. The use of cell-free aminopeptidases in place of an in vivo cellular source of the enzymes, although somewhat more cumbersome, it has the advantage of giving a more complete profile. Some amino-acid-βNAs are not transported into the cell for hydrolysis. Both methods produce different, although completely reproducible profiles.