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.

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu2' and Cd2+. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or P-mercaptoethanol, while Zn2+ or Co2' restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (Km, 0.55 mM) but that it can hydrolyze this substrate at a high rate (Vmax, 30 ,umol/min per mg of protein).

The nucleotide scqucncc of the pci~'I,N gene from toc/orocc.rt.s Iurrb erlcoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated ilf, of 95,368, which aprccs with the apparent M,of95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopcptidasc. The amino acid sequence of the aminopeptidase of L. fuctis wss found to be similar to the corresponding cnzymcs of human, rat and mouse, with host 30% of the residues identical. Also. a highly conserved arca was identified which has similarity with the uctivc site of thcrmolysin. A zinc-binding site, as well as the catalytic site for RpN, is predicted to lie within this conscrvcd stretch, Putative promoter regions upstream of PcpN were confirmed by primer extension analysis.

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anionexchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38°C. The enzyme is inactivated by the chemical agents 1,10phenanthroline, ethylenedinitrilotetraacetate, ,B-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2' and Zn2+. The ethylenedinitrilotetraacetateor 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacilus spp. and Streptococcus salivarius subsp. thermophilus. * Corresponding author. such activity. As expected, such activity was found, and this paper reports the purification and characterization of an endopeptidase from L. lactis subsp. cremoris Wg2. This new peptidase can hydrolyze large P-casein peptides into smaller fragments. This enzyme appears to be distinctly different from other reported endopeptidases (48, 49). MATERIALS AND METHODS Organisms and preparation of cell extract. L. lactis subsp. cremoris Wg2 was obtained from the Netherlands Institute for Dairy Research (NIZO), Ede, The Netherlands. Lactococcus lactis subsp. cremoris H61 was obtained from the National Institute of Animal Industry, Ibaraki, Japan; L. lactis subsp. cremoris P8-2-47 was obtained from the Institut fur Mikrobiologie, Bundesanstalt fuir Milchforschung, Kiel, Germany. L. lactis subsp. cremoris AM2, also called CNRZ 380, was obtained from the culture collection of the

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.