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Applied and Environmental Microbiology, February 2003, p. 1276-1282, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1276-1282.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Lactobacillus helveticus PepO2, an Endopeptidase with Post-Proline Specificity

Yo-Shen Chen,1 Jeffrey E. Christensen,2,{dagger} Jeffery R. Broadbent,3 and James L. Steele1*

Departments of Food Science,1 Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706,2 Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 843223

Received 8 April 2002/ Accepted 1 November 2002

A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-ß-casein-(f203-209)-{rho}-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as ß-casein(f193-209).

* Corresponding author. Mailing address: Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706. Phone: (608) 262-5960. Fax: (608) 262-6872. E-mail: jlsteele{at}facstaff.wisc.edu.

{dagger} Present address: Clinical Research Department, Marshfield Medical Research Foundation, Marshfield, WI 54449.

Applied and Environmental Microbiology, February 2003, p. 1276-1282, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1276-1282.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Cai, H., Thompson, R., Budinich, M. F., Broadbent, J. R., Steele, J. L. (2009). Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution. Gen Biol Evol 2009: 239-257 [Abstract] [Full Text]  
  • Janer, C., Arigoni, F., Lee, B. H., Pelaez, C., Requena, T. (2005). Enzymatic Ability of Bifidobacterium animalis subsp. lactis To Hydrolyze Milk Proteins: Identification and Characterization of Endopeptidase O. Appl. Environ. Microbiol. 71: 8460-8465 [Abstract] [Full Text]  
  • Sridhar, V. R., Hughes, J. E., Welker, D. L., Broadbent, J. R., Steele, J. L. (2005). Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides. Appl. Environ. Microbiol. 71: 3025-3032 [Abstract] [Full Text]  
  • Christensen, J. E., Broadbent, J. R., Steele, J. L. (2003). Hydrolysis of Casein-Derived Peptides {alpha}S1-Casein(f1-9) and {beta}-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase. Appl. Environ. Microbiol. 69: 1283-1286 [Abstract] [Full Text]  


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