The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides

Abstract

The peptides released from ␤-casein by the action of P I -type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of ␤-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this ␤-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH ؉ mass of each eluted peptide. All peptides corresponding to each of the MH ؉ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of ␤-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di-and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from ␤-casein by proteinase only.Lactococci have very limited capacities of synthesizing amino acids and therefore must utilize exogenous nitrogen sources for optimal growth. The amino acid requirement is strain dependent, but Glu or Gln, Ile, Leu, His, Met, and Val are essential for the growth of most Lactococcus lactis strains (6). The addition of several other amino acids was found to be growth stimulatory (34,25). The concentrations of essential amino acids in milk are very low, especially those of Ile and Leu (less than 1 mg/liter) (27). Moreover, the concentrations of other free amino acids are too low to explain the growth of L. lactis to the cell densities normally reached in coagulated milk (27,44). Thus, for optimal growth in milk, lactococci depend on the utilization of milk proteins, such as caseins. Casein hydrolysis by lactococci is mediated by a complex proteolytic system which includes a cell envelope-located proteinase (P I -or P III -type proteinase [PrtP]) and several peptidases (for recent reviews, see references 31, 32, and 42).According to proposed models, PrtP is involved in the first step of casein degradation (32, 39). The action of purified PrtP on ␤-casein has been studied extensively (28,29,33,46,47). After separation of the proteolytic products by reverse-phase high-performance liquid chromatography (HPLC), the different peptides are collected, purified when needed, and identified by biochemical methods (i.e., amino acid composition, determina...

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides

“…The central role of the oligopeptide transport system in the proteolytic pathway of L. lactis has recently been demonstrated (10). The need for di-and tripeptide transport systems in L. lactis is less apparent, since di-and tripeptides are not released from ␤-casein by the proteinase PrtP (9). However, growth of L. lactis in medium containing a mixture of caseins requires the presence of a functional di-and tripeptide transport system(s) (26).…”

“…However, growth of L. lactis in medium containing a mixture of caseins requires the presence of a functional di-and tripeptide transport system(s) (26). Although small peptides have not been detected in the degradation of ␤-casein by PrtP (9), it has been suggested that at least one essential amino acid is taken up in the form of a di-or a tripeptide which could be released from casein species other than the ␤ form (e.g., -casein) (20,23).…”

Utilisation des sources d'azote du lait par Lactococcus lactis

“…C'est également la caséine utilisée préfé-rentiellement lors de la croissance de L lacfis (Exterkate et de Veer, 1987). De ce fait, de nombreuses études ont porté sur l'identification des peptides libérés après hydrolyse de la caséine~par une protéase de paroi purifiée (Visser et al, 1988(Visser et al, , 1991Monnet et al, 1989 ;Reid et al, 1991 (Juillard et al, 1995b a From Tan et al, 1993. approche a prouvé que l'hydrolyse de la caséine p par une protéase de paroi génère un nombre de peptides beaucoup plus important que ce que l'on pensait jusqu'alors.…”

“…Enfin, la croissance dans du lait de ces différents mutants corrobore les résultats des expériences de transport. Le niveau cellu- Juillard et al, 1995b . laire atteint par une souche Prtt incapable de transloquer les oligopeptides représente moins de 5 % de celui atteint par la souche sauvage .…”

The specificity of oligopeptide transport by Streptococcus thermophilus resembles that of Lactococcus lactis and not that of pathogenic streptococci

“…lactocepin, which mainly releases peptides of large molecular mass from caseins (Juillard et al, 1995b;Kunji et al, 1996).…”