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Applied and Environmental Microbiology, June 2005, p. 3025-3032, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3025-3032.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

Vidya R. Sridhar,^1, Joanne E. Hughes,³ Dennis L. Welker,³ Jeffery R. Broadbent,² and James L. Steele^1*

Department of Food Science, University of Wisconsin—Madison, Madison, Wisconsin 53706,¹ Western Dairy Center and Department of Nutrition and Food Sciences,² Department of Biology, Utah State University, Logan, Utah 84322³

Received 27 July 2004/ Accepted 29 December 2004

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5 derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, ß-casein (ß-CN) (f193-209) and _S1-casein (_S1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards _S1-CN (f1-9) and ß-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze ß-CN (f193-209) and _S1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides _S1-CN (f1-9), _S1-CN (f1-13), and _S1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with ß-CN (f193-209) and _S1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.

Present address: 2730 S.W. Schiller Terrace, Portland, OR 97225.

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