The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.
A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.
Gobbetti
M
M
Institute of Dairy Microbiology, Agricultural Faculty of Perugia, Italy.
Smacchi
E
E
Corsetti
A
A
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004150:Dipeptidases / Q000302:isolation & purification* / Q000378:metabolism; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D010450:Endopeptidases; D000626:Aminopeptidases; D004150:Dipeptidases