Appl Microbiol Biotechnol (1989) 31:75-78 @plied ACwrobiology Biotechnology © Springer-Verlag 1989 Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp, cremoris Barbara Kiefer-Partsch, Wilhelm Boekelmann, Arnold Geis, and Michael Teuber Institut for Mikrobiologie, Bundesanstalt fiir Milchforschung, Hermann-Weigmann-Strasse 1, D-2300 Kiel 1, Federal Republic of Germany Summary. The//-casein specific cell wall proteo- lytic system of Lactococcus lactis subsp, cremoris P8-2-47 contains a metal-independent X-prolyl- dipeptidyl-aminopeptidase. Suitable substrates for its assay are Gly-Pro-nitroanilide and Ala-Pro- nitroanilide. It is suggested that the function of the enzyme is to cleave the proline-rich sequences of//-casein, as shown by the degradation of fl-ca- somorphin. It is a serine proteinase with a mono- mer molecular mass of about 90000 daltons, a temperature optimum of 45 °-50 ° C, and a pH op- timum of about 7. Introduction Lactococcus lactis subsp, cremoris P8-2-47 is able to grow in milk due to a fl-casein-degrading pro- teolytic system located in the cell wall (Thomas and Pritchard 1987). The first enzyme in the nec- essary cascade of reactions from the initial break- down of fl-casein to the transport of amino acids and peptides into the cytoplasm is a highly//-ca- sein-specific protease in most strains, called Lac- tococcus p-caseinase in this communication. This enzyme, which is usually coded for on plasmids (Kok et al. 1985), has an apparent molecular mass of 145 000 daltons (Geis et al. 1985). The nucleo- tide sequence of its gene has recently been estab- lished (Kok et al. 1988). The fl-casein peptides generated by the purified/%caseinase have been isolated and characterized. The acid soluble pep- tides of fl-casein contain between two and 24 am- ino acids (Monnet et al. 1986; Bockelmann 1987). The peptide bonds specifically split by the fl-ca- Offprint requests to: W. Bockelmann seinase are located between phenylalanine 52 and glycine 94, and glutamic acid 160 and glutamic acid 194 of the/%casein peptide chain. All these peptides disappear when incubated with intact cells of L. lactis subsp, cremoris P8-2-47. Since large peptides cannot be transported through the bacterial cell membrane, other enzymes found in the cell wall fractions, including a dipeptidase (van Boven et al. 1988), an aminopeptidase (Geis et al. 1985) and a membrane bound peptidase (Exterkate and de Veer 1987) must complement the /%caseinase. Using peptides isolated from a Lactococcus fl-caseinase digest of fl-casein and synthetic bovine//-casomorphin as substrates, we detected an additional metal-independent pepti- dase activity in the//-casein specific proteolytic system. Due to the discovered specificity for X- prolyl moieties, Gly-Pro-4-nitroanilide turned out to be an excellent model substrate for the charac- terization of this new enzyme. Materials and methods Bacteria and culture conditions. Lactococcus lactis subsp, cremo- ris P8-2-47 was isolated from a mesophilic dairy starter culture (Andresen et al. 1984). The organism was routinely maintained frozen in litmus milk at - 74 ° C. For the preparation of cells, M17 broth (Terzaghi and Sandine 1975) was inoculated with 1% bacteria, pregrown for several cycles in litmus milk and passaged once in M17 broth. Incubation was at 30°C for 16h. Isolation and purification procedure. Step 1 : peptidase activity was solubilized from the cells by repeated washings (2-3 times) of intact cells with 50 mM Tris-HC1 buffer, pH 7.5 (Geis et al. 1985); 20 g cells (wet weight) were suspended in 200 ml buffer, stirred for 30 min at 20 ° C and collected by centrifuga- tion. Step 2: anion exchange chromatography (Q-Sepharose Fast Flow, Pharmacia, Uppsala, Sweden); a linear gradient of 0-0.3 M NaC1 in 50 mM TRIS-HCI, pH 7.5 was used. Step 3 : chromatofocusing (Mono-P HR 5/20 column, Pharmacia); the