Characterization of the pattern of alphas1- and beta-casein breakdown and release of a bioactive peptide by a cell envelope proteinase from Lactobacillus delbrueckii subsp. lactis CRL 581.
The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter Lactobacillus delbrueckii subsp. lactis CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzyme's production. The carbon source and osmolites did not affect CEP activity. The CEP of L. delbrueckii subsp. lactis CRL 581 exhibited a mixed-type CEP(I/III) variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the alpha(s1)- and beta-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of alpha(s1)- and beta-casein breakdown was defined and is reported herein, this being the first report for a CEP of L. delbrueckii subsp. lactis. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized.
Hebert
Elvira María
EM
Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145, 4000 San Miguel de Tucumán, Argentina. ehebert@cerela.org.ar
Mamone
Gianfranco
G
Picariello
Gianluca
G
Raya
Raúl R
RR
Savoy
Graciela
G
Ferranti
Pasquale
P
Addeo
Francesco
F
D000595:Amino Acid Sequence; D002241:Carbohydrates; D002244:Carbon / Q000378:metabolism; D002364:Caseins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D048668:Lactobacillus delbrueckii / Q000201:enzymology* / Q000254:growth & development; D008055:Lipids; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014674:Plant Proteins, Dietary / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D002241:Carbohydrates; D002364:Caseins; D003470:Culture Media; D008055:Lipids; D010455:Peptides; D014674:Plant Proteins, Dietary; C080652:Osmolite; D002244:Carbon; D010447:Peptide Hydrolases