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...

Directly upstream of the Lactococcus lachs subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORFI was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking, ORFl were phenotypicaHly proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca2+-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca2+ inhibited, while ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca2+-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.

Activity of the lactococcal cell envelope-located serine proteinase depends on the presence of membraneassociated lipoprotein PrtM. To differentiate between the action of the proteinase and the action of PrtM in the process of proteinase maturation, an inactive form of the lactococcal proteinase was constructed. This was done by mutating one of the three amino acids thought to constitute the active site of the enzyme. The secreted form of this inactivated proteinase was the same size as the inactive secreted form of the proteinase produced in the absence of PrtM. Both inactive proteinases are larger than the active proteinase. Isolation of proteinase by washing lactococcal cells carrying the complete proteinase gene in a Ca2"-free buffer was prevented by the

No abstract

The Streptococcus cremoris Wg2 proteinase gene, cloned in S. lactis, specified a proteinase which exhibited the same specificity toward casein as did the proteinase isolated from the original host. Although the cloned gene lacked the last 130 codons, the proteinase still specifically degraded I-casein. Deletion of the C-terminal 343 amino acids from the proteinase did not influence this specificity. Cell-free transcription-translation studies of plasmids carrying deletion derivatives of the proteinase gene showed that the 100-kilodalton C-terminally truncated proteinase still exhibited proteolytic activity. Crossed immunoelectrophoresis revealed that proteins A and B identified in the proteolytic system of S. cremoris Wg2 are both encoded by the proteinase gene. A working model based on integration of available genetic, immunological, and biochemical data is presented to explain this result.

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a p of 0.23 h-I. At higher growth rates the proteinase production declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. Iuctis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat.The addition of the dipeptide leucylproline (final concentration of 100 pM) to a lactose-limited continuous culture during the steady state (D = 0.23 h-I) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. luctis.

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.