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Abstract
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.
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pI (isoelectric point)References
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Article info
Publication history
Accepted:
January 31,
1997
Received:
April 18,
1996
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© 1997 American Dairy Science Association. Published by Elsevier Inc.
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