Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine asl-, 13-, and K-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM1, was concluded. The action of the HP-type proteinase PI (also detectable in strains Wg2, C13, and TR) was established by electrophoretic methods to be directed preferentially towards 13-casein. The AM,-type proteinase Pm (also detectable in strain SK11) was also able to degrade 1-casein, but at the same time split asl-and K-casein more extensively than did PI. Strain FD27 exhibited mainly PI activity but also detectable Pm degradation characteristics. The cell wall proteinase preparation of strain E8 showed low PI as well as low Pm activity. All proteinase preparations produced from K-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between PI and Pm in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-14C-labeled (-casein and by the effect of asl-plus K-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.

No abstract

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that 13-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. K-casein hydrolysis is a relatively slow process, and os-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using 14CH3-labeled caseins. A relatively fast and linear initial progress of 14CH3-labeled 13-casein degradation is not inhibited by ae-casein and only slightly by K-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of ,I-casein degradation for growth of the organism in milk are discussed.

A membrane-bound L-a-glutamyl (aspartyl)-peptide hydrolase (aminopeptidase A) (EC 3.4.11.7) from Streptococcus cremoris HP has been purified to homogeneity. The free y-carboxyl group rather than the amino group of the N-terminal L-a-glutamyl (aspartyl) residue appeared to be essential for catalysis. No endopeptidase activity could be established with this enzyme. The native enzyme is a polymeric, most probably trimeric, metalloenzyme (relative molecular weight, approximately 130,000) which shows on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels apparent high relative molecular weight values due to (lipid?) material dissociable with butanol. The subunit (relative molecular weight, approximately 43,000) is catalytically inactive. The enzyme is inactivated completely by dithiothreitol, chelating agents, and the bivalent metal ions Cu2'and Hg2+. Of the sulfhydryl-blocking reagents tested, only p-hydroxymercuribenzoate appeared to inhibit the enzyme. Activity lost by treatment with a chelating agent could be restored by Co2+ and Zn2+. The importance of the occurrence of an aminopeptidase A in S. cremoris with respect to growth in milk is discussed.

No abstract