SUMMARYA total of 169Lactobacillusstrains from 12 species (Lb. acidophilus, Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckiisubsp.bulgaricus, Lb. delbrueckiisubsp.delbrueckii, Lb. delbrueckiisubsp.lactis, Lb. fermentum, Lb. helveticus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. rhamnosus), isolated from raw milk and various milk products, and 9Lactococcus lactisstrains were evaluated for peptidase activities with five chromogenic substrates and a tryptic digest of casein. Within each species, the peptidase activity of the cell-free extracts of the strains varied. Furthermore, differences were observed between theLactobacilhisspecies andLc. lactis. Lb. helveticushad by far the highest hydrolysing activities towards all substrates, indicating the presence of powerful aminopeptidases, X-prolyl-dipeptidyl aminopeptidases and proline iminopeptidases.Lb. delbrueckiisubsp.bulgaricuspossessed high hydrolysing activities towards substrates containing proline, alanylprolyl–p–nitroanilide and prolyl–p–nitroanilide. On the other hand,Lb. fermentumandLb. breviscould be considered as weakly proteolytic species. A more detailed study with highly proteolyticLactobacillusstrains indicated that at least three different proteinases or endopeptidases were present. Compared withLc. lactis, theLactobacillusstrains had a much lower hydrolytic action on glutamyl-glutamic acid, suggesting that glutamyl aminopeptidase was absent in lactobacilli.

The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:1460-1468. The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase. The steady-state levels of pathway intermediates upon refeeding with lactose after various periods of starvation indicate that the decreased glycolysis is primarily due to diminished glyceraldehyde-3-phosphate dehydrogenase activity. Furthermore, quantification of the control strength exerted by glyceraldehyde-3-phosphate dehydrogenase on the overall activity of the glycolytic pathway shows that this enzyme can be significantly rate limiting in nongrowing cells.In a previous communication, we have presented evidence that the survival of Streptococcus cremoris upon lactose starvation in chemostat cultures is related to the glycolytic capacity maintained (12). A rapid decrease of glycolytic activity was observed for lactose-starved cells in growth medium (anaerobically) and in phosphate-lactate buffer (aerobically) (12). Initially, the decrease of glycolysis did not result in a lower steady-state level of the proton motive force (Ap), but the rate of proton motive force generation was severely affected. However, when glycolysis decreased further over time, the high proton motive force could not be maintained and the activity of proton motive force-driven processes, like amino acid transport, decreased (12). In this paper, the cause of the drop in glycolytic activity upon (lactose) starvation has been analyzed.Enzymatic activities. The decrease in glycolysis during starvation as described previously (12) was also observed for S. cremoris Wg2 (and S. lactis ML3) cells grown on complex broth (4) in batch cultures (Fig. 1). Cells were grown to an optical density at 660 nm of about 0.8, washed twice, and suspended in sterile phosphate-lactate buffer (pH 6.4) as described elsewhere (12). At the times indicated, samples were taken and the acidification of the medium was monitored (11). To study the effect of starvation on the activities of glycolytic enzymes, cell-free extracts were prepared (10) after three different periods of starvation, as indicated by the arrows (Fig. 1). Enzymatic activities were determined by standard procedures involving NAD, NADH-, or NADPcoupled assays (3). The effects of starvation on the activites of glyceraldehyde-3-phosphate (G3P) dehydrogenase (EC 1.2.1.12) and phosphoglycerate mutase (EC 5.4.2.1) are shown in Table 1. Both activities decreased concomitant with the decrease in glycolysis. The activities of all other glycolytic enzymes were not significantly affected by the starvation regime (data not shown). The activities of these enzymes varied between 1 and 6 U/mg of protein, except for phosphoglycerate kinase (EC 2.7.2.3), which had an activity of about 0.4 U/mg of protein, independent of the starvation ...

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremonis Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55°C. EDTA inhibits the activity, and this can be reactivated with Zn2+, Mn2+, and partially with Co2+. The reducing agents dithiothreitol and 13-mercaptoethanol and the divalent cation Cu21 inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a Km of 0.15 mM and a Vm. of 151 ,umol/min per mg of protein.

An aminopeptidase with a very broad substrate specificity was purified to homogeneity from Lactobacillus helveticus SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide-and nitroanilide-substituted amino acids, as well as several di-, tri-and oligopeptides. It also exhibited significant prolineiminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a Vmex of 2.5 pmol min-l (mg protein)-' while lysyl-p-nitroanilide was hydrolysed with a high affinity [K, 0003 mM; VmaX 37.5 pmol min-l (mg protein)-l]. The aminopeptidase activity, which was optimal between pH 6.0 and 8 0 and at 50 "C, was very stable a t 30 "C for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with P-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are a t least immunologically different from each other.

SUMMARYCell extracts of various lactobacilli and twoLactococcusstrains were investigated for their immunoresponse with monoclonal and polyclonal antibodies raised against various proteolytic enzymes fromLc. lactis. Except forLactobacillus caseiSBT 2233, none of the lactobacilli proteins showed immunoresponse with the monoclonal antibodies. With polyclonal antibodies raised against aminopeptidases N and C and endopeptidase ofLc. lactisan immunoresponse was observed. However, the molecular masses of the reactive bands on the blot were considerably different from those of the corresponding lactococcal peptidases, except for the band that reacted with polyclonal antibodies against aminopeptidase C. The polyclonal antibodies raised against X-prolyl-dipeptidyl aminopeptidase and tripeptidase did not show any immunoreaction. As a control, all antibodies reacted with the lactococcal proteins on the blot, with molecular masses corresponding to those reported for the proteinases and peptidases. The results clearly showed that most of the proteolytic enzymes of lactobacilli were immunologically different from those of lactococci. The proteolytic enzymes in the cell-free extracts were separated by non-denaturing PAGE and visualized by zymogram staining. The electrophoretic pattern of the proteolytic enzymes of lactobacilli was different from that ofLc. lactis. Both experiments indicate that the enzymes of the proteolytic system of lactobacilli are different from those of lactococci.