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

¹ Instituto del Frio (CSIC), Ciudad Universitaria s/n, 28040 Madrid, Spain

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.

Key Words: Lactobacillus case • dipeptidase • purification • characterization

Submitted on April 18, 1996
Accepted on January 31, 1997

This article has been cited by other articles:

				A. Matthews, A. Grimaldi, M. Walker, E. Bartowsky, P. Grbin, and V. Jiranek Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification Appl. Envir. Microbiol., October 1, 2004; 70(10): 5715 - 5731. [Full Text] [PDF]

				P. Varmanen, T. Rantanen, A. Palva, and S. Tynkkynen Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus Appl. Envir. Microbiol., May 1, 1998; 64(5): 1831 - 1836. [Abstract] [Full Text]