Kappacin, a novel antibacterial peptide from bovine milk

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa -casein composed of glycosylated and phosp horylated forms of different genetic variants. We have demonstrated that CM P has growth-inhibitory activity against the oral opportunistic pathogens S treptococcus mutans and Porphyromonas gingivalis and against Escherichia co li. CMP was fractionated using reversed-phase highperformance liquid chroma tography (RP-HPLC), and each fraction was tested for activity against S. mu tans in a 96-well-plate broth assay. Fractions were characterized by N-term inal sequence analysis and mass spectrometry. The active form of CMP was sh own to be the nonglycosylated, phosphorylated kappa -casein (residues 106 t o 169) [kappa -casein(106-169)], which we have designated kappacin. Endopro teinase Glu-C was used to hydrolyze CMP, and the generated peptides were se parated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa -casein-A(138-158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited gro wth-inhibitory activity. Peptides corresponding to the sequences of the inh ibitory peptide Ser(P)(149)kappa -casein-A(138-158) and its nonphosphorylat ed counterpart kappa -casein-A(138-158) were chemically synthesized and tes ted for antibacterial activity. The synthetic Ser(P)(149)kappa -casein-A(13 8-158) displayed growth-inhibitory activity against S. mutans (MIC, 59 mug/ ml [26 muM]). The nonphosphorylated peptide, however, did not inhibit growt h at the concentrations tested, indicating that phosphorylation is essentia l for activity.