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Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides.[Erratum: 2005 July, v. 71, no. 7, p. 4161.]

Author:
Sridhar, Vidya R., Hughes, Joanne E., Welker, Dennis L., Broadbent, Jeffery R., Steele, James L.
Source:
Applied and environmental microbiology 2005 v.71 no.6 pp. 3025-3032
ISSN:
0099-2240
Subject:
Lactobacillus helveticus, cheese starters, proteinases, genes, genome, nucleotide sequences, peptides, bitter-tasting compounds, enzymatic hydrolysis, enzyme activity, gene transfer, genetic transformation, gene expression, Lactococcus lactis, Cheddar cheese, cheese ripening, bitterness
Abstract:
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[alpha] derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, {szligbeta}-casein ({szligbeta}-CN) (f193-209) and [alpha][subscript S1]-casein ([alpha][subscript 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 [alpha][subscript S1]-CN (f1-9) and {szligbeta}-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 {szligbeta}-CN (f193-209) and [alpha][subscript S1]-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides [alpha][subscript S1]-CN (f1-9), [alpha][subscript S1]-CN (f1-13), and [alpha][subscript 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 {szligbeta}-CN (f193-209) and [alpha][subscript 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.
Agid:
1410827