Proteolytic Specificity of Chymosin on Bovine α_sl-Casein

The proteolytic specificity of chymosin (EC 3.4.23.4) on bovine α_s1- casein at 30 °C in phosphate buffer, pH 6.5 and at pH 5.2 in the presence of 5 % (w/v) NaCl was investigated. Peptides (pH 4.6-soluble) were isolated by reversed-phase HPLC and identified from their amino acid sequence; the identity of some peptides was confirmed by mass spectrometry and/or amino acid composition. The small peptides produced at pH 6.5 were Arg₁-Phe₂₃ Phe₂₄-Phe₂₈ Phe₂₄-Leu₄₀(?), Phe,5₀-Phe₁₅₃ Phe₁₅₀-Leu₁₅₆ Tyr₁₅₄-Tyr₁₅₉ Tyr₁₆₄-Trp₁₆₄ Asp₁₅₇-Trp₁₆₄ and Tyr₁₆₅-Trp₁₉₉. The same peptides, except Tyr₁₅₄-Trp₁₆₄ were produced at pH 5.2 in the presence of NaCl and, in addition, the peptides Arg₁-Leu₁₁ Phe₂₄-Phe₃₂ Lys₁₀₂-Leu₁₄₂ Ala₁₄₃-Leu₁₄₉ and Tyr₁₆₅-Phe₁₇₉. The rates of production of individual peptides differed under the two conditions studied but ArgPhe and Tyr₁₆₅-Trp₁₉₉ were the first and second peptides produced under both conditions. Pathways are proposed to interpret the proteolysis of α_sl-casein in solution under the conditions of this study.