Three enzymes with Land one enzyme with D-aminopeptidase (EC 3.4.11; a-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W. T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 i 20,000, is produced early in growth, and hydrolyzes L-alanyl- ,-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 i 10,000, is also produced early in growth and hydrolyzes L-lysyl-,@-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 i 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-a-aspartyl- ,-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-Dalanyl-B-naphthylamide (as well as D-alanyl-,B-naphthylamide and D-alanyl-Dalanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 1 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism.
