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Applied and Environmental Microbiology, December 2005, p. 8460-8465, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8460-8465.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Enzymatic Ability of Bifidobacterium animalis subsp. lactis To Hydrolyze Milk Proteins: Identification and Characterization of Endopeptidase O

C. Janer,1 F. Arigoni,2 B. H. Lee,3 C. Peláez,1 and T. Requena1*

Departamento de Ciencia y Tecnología de Productos Lácteos, Instituto del Frío (CSIC), José Antonio Novais 10, Madrid 28040, Spain,1 Microbiology Unit, Nestlé Research Center, Vers-Chez-les-Blanc, Lausanne 1000, Switzerland,2 Department of Food Science and Agricultural Chemistry, McGill University/AAFC, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V93

Received 16 May 2005/ Accepted 6 September 2005

The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide {alpha}s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.

* Corresponding author. Mailing address: Departamento de Ciencia y Tecnología de Productos Lácteos, Instituto del Frío (CSIC), José Antonio Novais, 10, 28040 Madrid, Spain. Phone: (34) 91 5492300. Fax: (34) 91 5493627. E-mail: trequena{at}if.csic.es.

Applied and Environmental Microbiology, December 2005, p. 8460-8465, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8460-8465.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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  • Seo, J. M., Ji, G. E., Cho, S. H., Park, M. S., Lee, H. J. (2007). Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family. Appl. Environ. Microbiol. 73: 5598-5606 [Abstract] [Full Text]  


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