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 degrees 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.
Fernandez-Espla
M D
MD
Instituto del Frio (CSIC), Madrid, Spain.
Martin-Hernandez
M C
MC
D000818:Animals; D002611:Cheese / Q000382:microbiology*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004150:Dipeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006041:Goats; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity
D004150:Dipeptidases; C038937:dipeptidase