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-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 degrees C. The enzyme was incubated at 40 degrees C for 15 min with various metal ions. It was activated by Mg (2.5 mM), Ca (0.1 to 2.5 mM), Na (10 to 50 mM), and K (10 to 50 mM) and was inhibited by Hg (0.1 to 2.5 mM), Cu (0.1 to 2.5 mM), and Zn (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40 degrees C), 1,10-phenanthroline (1.0 mM, 15 min at 40 degrees 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 degrees C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40 degrees C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).
Khalid
N M
NM
Department of Food Science and The Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706.
Marth
E H
EH