PepS from Streptococcus thermophilus

International audienc

Purification and Characterization of a Dipeptidase from Lactobacillus casei ssp. casei IFPL 731 Isolated from Goat Cheese Made from Raw Milk

A dipeptidase was purified to homogeneity from the cell-free extract of Lactobacillus casei ssp. casei IFPL 731 by a combination of heat treatment, hydrophobic interaction chromatography, anion-exchange chromatography, and gel filtration. A purification factor of 395-fold was obtained, and yield was 20%. The dipeptidase was shown to be a metal-dependent enzyme; optimal activity was at pH 7.5 and 60 to 75°C, and the enzyme had a high degree of thermal stability. Molecular mass was estimated by SDS-PAGE and gel filtration to be 46 kDa, which suggested that the enzyme existed as a monomer. Enzyme activity was most effectively inhibited by metal-chelating agents, reducing agents, or sulfhydryl group reagents. After inhibition with phenanthroline, activity was partially restored by Co2+ and Mn2+. The kinetics of Phe-Ala and Leu-Leu did not follow Michaelis-Menten saturation kinetics but exhibited a mixture of positive and negative cooperativity for the successive binding of molecules of the same substrate.This work was carried out under the terms of reference of research projects ALI94-0735 (Interministerial Commission for Science and Technology) and AAIR 2-CT93-1531.Peer Reviewe

Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization

International audienc

Purification and Characterization of a Novel Serine Aminopeptidase from Lactobacillus casei Ssp. casei IFPL 731

An aminopeptidase showing broad specificity has been purified to homogeneity from the cell-free extract of Lactobacillus casei ssp. casei IFPL 731. Enzyme activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride, and reducing agents such as dithiothreitol and β-mercaptoethanol. The metal chelating agent, ethylenediamintetraacetic acid, also reduced enzyme activity. The molecular mass of the purified enzyme was estimated to be 67 kDa by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme exits as a monomer. The purified enzyme hydrolyzed p-nitroanilides of several amino acids and peptides as well as di- and tripeptides. The best substrates were Arg-Pro-p-nitroanilide, Ala-Pro-p-nitroanilide, Phe-Met, Leu-Gly, Phe-Ala, and Leu-Gly-Phe. Km values for Arg-Pro-p-nitroanilide and Leu-Gly were 4.8 and 1.1 mM, respectively. The properties of the enzyme are compared with those of other aminopeptidases isolated from lactic acid bacteria.Peer Reviewe