Isolation and physical characterization of an aminopeptidase from culture filtrates of Bacillus licheniformis (Weigmann)

Abstract

An aminopeptidase was purified 470 fold from culture filtrates of Bacillus licheniformis. The enzyme behaved as a homogeneous protein when it was subjected to disc gel electrophoresis and sedimentation velocity. The zinc to protein ratio of the fractions obtained increased 34 fold during the purification procedure, thus indicating that the enzyme contains zinc. The minimum molecular weight calculated from the zinc content of the purified aminopeptidase was 40,350 a value in close agreement with those found by sedimentation equilibrium (41,700) and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (37,000). Thus, zinc was present in the stoichiometric ratio of one gram atom per mole of the enzyme. The enzyme consists of a single polypeptide chain, as evidenced by the results of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol. Isoelectric focusing experiments revealed that the isoelectric pH of the aminopeptidase was 4.96 ± 0.09. The sedimentation coefficient reduced to standard conditions (20°, w) was 3.83 S and an estimated value for the diffusion coefficient at standard conditions was 8.01 x10�� cm² sec�¹. Experiments designed to determine substrate specificity indicated that the enzyme hydrolyzes amino acid amides, dipeptides and aminoacyl- β- naphthylamides; however, in all cases a free N-terminal amino group of the L configuration was required. Optical rotatory dispersion measurements showed that the enzyme exhibits a negative Cotton effect at 233 m μ. The optimum pH for enzymatic activity was found to be close to 8.3 in 0.082 sodium barbital buffer at r/2=0.10. The Km values for leucyl- β-naphtylamide and leucyl-p-nitroanilide were 5.1 x 10�� and 2.86 x 10�� moles per liter respectively.