A dimeric, extracellular, heat-stable aminopeptidase produced by a marine pseudomonad

Abstract

An extracellular aminopeptidase (Alteromonas aminopeptidase III) of a marine pseudomonad, designated Alteromonas B-207, was purified to homogeneity. The enzyme was found to have a native molecular weight of 63 000 by exclusion chromatography and a subunit weight of 33 000 by SDS-polyacrylamide gel electrophoresis. Cross-linking with dimethyladipimidate prevented dissociation of the dimer. The enzyme has a pH optimum of 9.3, a temperature optimum of 70°C and is stable at 60°C for approx. 1 h. It has high specificity for l-leucyl-β-naphthylamide and, of the peptides tested, it shows a preference for l-Leu, Gly and l-Pro over l-α-Asp and l-Lys. The enzyme is inhibited by 1,10-phenanthroline, added to the enzyme-substrate reaction mixture, and by EDTA when the enzyme is dialyzed against 10⁻³ M soln. Activity can be partially restored to EDTA-dialyzed enzyme by removal of the EDTA and incubation of the enzyme with Zn²⁺. Atomic absorption data also implicate zinc as a required metal. Sulfhydryl- and serine- inhibitors have no effect on the enzyme.