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...

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Bibliographic Details
Published in:Journal of dairy science 1997-08, Vol.80 (8), p.1497-1504
Main Authors: Fernandez-Espla, M.D., Martin-Hernandez, M.C.
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Animals
CATION
CATIONES
CATIONS
characterization
CHEESE
Cheese - microbiology
CHEMICALS
Chromatography
Chromatography, Gel
Chromatography, Ion Exchange
dipeptidase
Dipeptidases - chemistry
Dipeptidases - isolation & purification
Dipeptidases - metabolism
Enzyme Stability
ENZYMIC ACTIVITY
FROMAGE
GOAT MILK
Goats
Hot Temperature
Hydrogen-Ion Concentration
Isoelectric Point
Kinetics
LACTOBACILLUS CASEI
Lactobacillus casei - enzymology
LACTOBACILLUS CASEI SUBSP. CASEI
LAIT DE CHEVRE
LECHE DE CABRA
METAL
METALES
METALS
MOLECULAR WEIGHT
PEPTIDASAS
PEPTIDASE
PEPTIDASES
PESO MOLECULAR
POIDS MOLECULAIRE
PRODUCTOS QUIMICOS
PRODUIT CHIMIQUE
PURIFICACION
PURIFICATION
QUESO
Substrate Specificity
TEMPERATURA
TEMPERATURE
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Summary: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 metaldependent 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.
ISSN:0022-0302
1525-3198
DOI:10.3168/jds.S0022-0302(97)76078-8