Purification and partial characterization of a prolyl-dipeptidyl aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-proplyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40 degrees C. The enzyme was incubated at 40 degrees C for 15 min with various metal ions. It was activated by Mg2+ (2.5 millimoles), Ca2+ (0.1 to 2.5 millimoles), Na+ (10 to 50 millimoles), and K+ (10 to 50 millimoles) and was inhibited by Hg2+ (0.1 to 2.5 millimoles), Cu2+ (0.1 to 2.5 millimoles), and Zn2+ (0.1 to 2.5 millimoles). Enzyme activity was partially inhibited by EDTA (1.0 millimoles, 20 h at 40 degrees C), 1,10-phenanthroline (1.0 millimoles, 15 min at 40 degrees C), phenylmethylsulfonyl fluoride (1.0 millimoles), N-ethylmaleimide (1.0 millimoles), and iodoacetate (1.0 millimoles). It was completely inhibited by diisopropyl fluorophosphate (1.0 millimoles, 2 h at 40 degrees C) and p-chloromercuribenzoate (1.0 millimoles, 15 min at 40 degrees C). The enzyme was not affected by dithioerythritol (1.0 to 10 millimoles)