PURIFICATION AND CHARACTERIZATION OF AMINOPEPTIDASE FROM LACTOBACILLUS-CASEI SSP CASEI LLG

An aminopeptidase of broad specificity was purified to homogeneity from Lactobacillus casei ssp. casei LLG by fast protein liquid chromatography. The enzyme was purified to 153-fold over the crude extract with a recovery of 9%. The purified enzyme appeared as a single band on native PAGE and SDS-PAGE and had a molecular weight of 87 kDa and an isoelectric point of 4.73. Purity of the enzyme was confirmed by the activity staining on substrate-coated blotted membrane. The enzyme hydrolyzed a range of nitroanilide-substituted amino acids, as well as dipeptides, and accounted for all of the aminopeptidase and some of the dipeptidase activities found in cell-free extracts. Kinetic studies indicated that the enzyme has a low affinity for leucine p-nitroanilide (Michaelis-Menton constant, .22 mM) but could hydrolyze the substrate at high rates (maximal velocity, 16.3 mmol/min per mg of protein). The aminopeptidase was shown to be a metal-dependent enzyme, because both EDTA and 1,10-phenanthroline caused complete inhibition of the aminopeptidase activity. Although Co2+ and Ni2+ could partially restore the activity lost by the treatment of 1,10-phenanthroline, EDTA caused an irreversible inhibition of the enzyme activity. Higher concentration of substrate and hydrolysis products also inhibited the activity of the enzyme. The enzyme showed optimal activity at pH 7.0 and at 39-degrees-C and was fairly stable over the pH range of 5 to 9 and below 45-degrees-C.