Pure subcellular fractions were obtained from a Lactobacillus casei strain, selected among numerous isolates from Grana cheese on the basis of their peptidolytic activity. Several lytic conditions for the attainement of stable protoplasts were checked and three different cellular markers were employed to assess subcellular fraction purity. The maximum yeld and the best stability of protoplasts. as followed by Scanning Electron Microscopy, were obtained using 25 mM Hepes pH 7, 15 mM MgCl2 and 20 U mutanolysin/mL, without the addition of osmotic stabilizers. Peptidolytic activities determined on subcellular fractions against Leu, Arg, Pro and Arg-Pro p-nitroanilides, had a cytoplasmic location. Comparative tests performed on either intact or broken cells by polyclonal antibodies against a general aminopeptidase and a X-prolyl dipeptidyl aminopeptidase confirmed this result. On the basis of such enzymatic activities, determined either on broken or intact cells, it is suggested that chromogenic substrates must be transported inside the cells.
