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Applied and Environmental Microbiology, April 2001, p. 1815-1820, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1815-1820.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

Yolanda Sanz* and Fidel Toldrá

Instituto de Agroquímica y Tecnología de Alimentos (CSIC), 46100 Burjasot (Valencia), Spain

Received 6 October 2000/Accepted 18 January 2001

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 µM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 µM, respectively. Among peptides, beta -casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.

* Corresponding author. Mailing address: Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Apartado 73, 46100 Burjasot (Valencia), Spain. Phone: 34 96 3900022. Fax: 34 96 3636301. E-mail: yolsanz{at}iata.csic.es.

Applied and Environmental Microbiology, April 2001, p. 1815-1820, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1815-1820.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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