THE EXTRACELLULAR P-I-TYPE PROTEINASE OF LACTOCOCCUS-LACTIS HYDROLYZES BETA-CASEIN INTO MORE THAN 100 DIFFERENT OLIGOPEPTIDES

The peptides released from beta-casein by the action of P-I-type prote inase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been ide ntified by on-line coupling of liquid chromatography to mass spectrome try. After 24 h of incubation of beta-casein with purified PrtP, a sta ble mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-pe rformance liquid chromatography and introduced into the liquid chromat ography-ion spray mass spectrometry interface. Multiply charged ions w ere generated from trifluoroacetic acid-soluble peptides under low noz zle voltage conditions, yielding the MR(+) mass of each eluted peptide : All peptides corresponding to each of the MH(+) calculated masses we re determined. In those cases in which different peptides were possibl e, further identification was achieved by collision-induced dissociati on under higher nozzle voltage conditions. Hydrolysis of beta-casein b y PrtP was observed to proceed much further than reported previously, More than 40% of the peptide bonds are cleaved by PrtP, resulting in t he formation of more than 100 different oligopeptides. With the except ion of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified olig opeptides are small enough to be taken up by the oligopeptide transpor t system. Uptake of these peptides could supply L. lactis with all ami no acids, including the essential ones, indicating that growth of L. l actis might be possible on peptides released from beta-casein by prote inase only.