SPECIFICITY OF HYDROLYSIS OF BOVINE KAPPA-CASEIN BY CELL ENVELOPE-ASSOCIATED PROTEINASES FROM LACTOCOCCUS-LACTIS STRAINS

The cell envelope-associated proteinases from Lactococcus lactis subsp . cremoris H2 (a P-I-type proteinase-producing strain) and SK11 (a P-I II-type proteinase-producing strain) both actively hydrolyze the kappa -casein component of bovine milk but with significant differences in t he specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Ly s-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the re lative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteina ses on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa- casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the b ond Glu-151-Val-152 were very rapidly cleaved by the P-III-type protei nase, whereas hydrolysis of these bonds by the P-I-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrol ysis of kappa-casein at these sites by the two different proteinase ty pes resulted in the formation of distinctive, high-M(r) products detec table by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Th e kappa-casein cleavage patterns revealed by sodium dodecyl sulfate-po lyacrylamide gel electrophoresis analysis provide a diagnostic basis f or recognizing differences in proteinase specificity and have been use d to identify P-I- or P-III-type and P-I/P-III intermediate-type prote inases from several different lactococcal strains.