Abstract
In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.
Highlights
The experiments described in this study have revealed a number of important properties of the proteolytic pathway of L. lactis
All of the essential and growth-stimulating amino acids for L. lactis can be released from J3-casein by the action of the proteinase PrtP in a form that can be transported by the cells
Since di-tripeptide transport mutants selected on the basis of resistance toward L-Ala-J3-chloro-L-Ala are affected in their ability to grow on a mixture of caseins (Smid et al, 1989b), we speculate that this is due to the inability to transport essential amino acids in the form of small peptides that are released from proteins other than J3-casein
Summary
Biochemical and immunological data indicate that the aminopeptidases (PepN and PepC), the Xprolyl-dipeptidyl aminopeptidase (PepX), the endopeptidase (PepO), the tripeptidase (Pep'I'), and the glutamyl aminopeptidase (PepA) are present inside the cell (Tan et al, 1992; Baankreis, 1992). In view of these observations, it would seem that the casein-derived peptides have to be taken up by the cells before further hydrolysis can take place. None ofthe amino acids accumulated significantly inside the cell when the Opp system was inactivated These observations provide direct evidence that peptidases are not involved in extracellular degradation and subsequent utilization of f3-casein by L. lactis
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