Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides.
The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.
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Institut National de la Recherche Agronomique, Laboratoire de Recherches de Technologie Laitière, 35042 Rennes Cédex, France.
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Gagnaire
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Atlan
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Lortal
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D000595:Amino Acid Sequence; D002364:Caseins / Q000235:genetics / Q000378:metabolism*; D002611:Cheese / Q000382:microbiology; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D013291:Streptococcus / Q000201:enzymology*
D002364:Caseins; D010446:Peptide Fragments; D010447:Peptide Hydrolases