DETECTION AND LOCALIZATION OF PEPTIDASES IN LACTOCOCCUS-LACTIS WITH MONOCLONAL-ANTIBODIES

Monoclonal antibodies against peptidases of Lactococcus lactis were is olated and characterized: PEPN1-4 against a lysyl aminopeptidase PepN, PEPT1-5 against a tripeptidase PepT and PEPD1-3 against a dipeptidase PepD. These monoclonal antibodies reacted specifically with their res pective antigens in crude cell extracts of Lc. lactis subspp. cremoris and lactis. A number of monoclonal antibodies cross reacted with prot eins of other (lactic acid) bacteria. PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa. protein in Lactobacillus delbrueckii, while PEPT 1 and PEPT2 reacted with proteins in the cell-free extract of Streptoc occus thermophilus and Clostridium fervidus. Of the four isolated mono clonal antibodies against PepN, only PEPN3 cross reacted weakly ,with a 90 kDa protein in Escherichia coli cell-free extract, and the other three antibody species against PepN cross reacted with 80 kDa proteins of Lb. casei, Lb. delbrueckii, and Str. bovis, but not of Esch. coli. Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins of Lb. casei, Lb. delbrueckii and Str. bovis. All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven different Lc. lactis strains, indicat ing that the peptidases of these strains were very similar to those of Lc. lactis subsp. cremoris WG2. However, Lc. lactis subsp. hordniae a ppeared to differ from the other Lc. lactis subspecies since only PEPT 1, 2 and 5 reacted with a protein in the cell-free extract. Immunogold labelling of Lc. lactis WG2 with the isolated monoclonal antibodies r evealed that PepN, PepD and PepT were located intracellularly. The int racellular location of these peptidases is discussed in relation to th e supply of essential amino acids and peptides.