Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.
A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.
Sahlstrøm
S
S
Department of Dairy and Food Industries, Agricultural University of Norway, As.
Chrzanowska
J
J
Sørhaug
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent / Q000494:pharmacology; D002473:Cell Wall / Q000201:enzymology; D002845:Chromatography; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D002413:Cations, Divalent; D009842:Oligopeptides; D011480:Protease Inhibitors; D010447:Peptide Hydrolases