Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

Abstract

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-prolyl-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�C. The enzyme was incubated at 40�C for 15 min with various metal ions. It was activated by Mg 2+ (2.5 mM), Ca 2+ (0.1 to 2.5 mM), Na + (10 to 50 mM), and K + (10 to 50 mM) and was inhibited by Hg 2+ (0.1 to 2.5 mM), Cu 2+ (0.1 to 2.5 mM), and Zn 2+ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40�C), 1,10-phenanthroline (1.0 mM, 15 min at 40�C), phenylmethylsulfonyl fluoride (1.0 mM), N -ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40�C) and p -chloromercuribenzoate (1.0 mM, 15 min at 40�C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).