Caseinolytic Specificity of Cardosin, an Aspartic Protease from the CardoonCynara cardunculusL.: Action on Bovine αs- and β-Casein and Comparison with Chymosin

The action of cardosin on bovine α s - and β-casein at 30 °C in 50 mM citrate buffer (pH 6.2) was studied. Peptides were isolated by reversed-phase HPLC on C 18 columns and identified from their amino acid composition and N -terminal amino acid sequence. The relative susceptibility of peptide bonds cleaved was Phe 23 -Phe 24 > Trp 164 -Tyr 165 > Tyr 166 -Val 167 > Tyr 165 -Tyr 166 > Phe 153 -Tyr 154 > Phe 145 -Tyr 146 ≈ Leu 149 -Phe 150 ≈ Leu 156 -Asp 157 ≈ Ala 163 -Trp 164 for α s1 -casein and Leu 192 -Tyr 193 > Leu 191 -Leu 192 ≈ Leu 165 -Ser 166 > Phe 190 -Leu 191 ≥ Ala 189 -Phe 190 ≈ Leu 127 -Thr 128 for β-casein. In α s2 -casein, cardosin cleaved the bonds Phe 88 -Tyr 89 and Tyr 95 -Leu 96 . The enzyme shows a clear preference for bonds between hydrophobic, bulky amino acids, cleaving four consecutive peptide bonds in extremely bulky, hydrophobic regions of both α s1 -CN (Ala 163 -Val 167 ) and β-CN (Ala 189 -Tyr 193 ), which was less attacked by chymosin in various experimental conditions. The active site cleft of cardosin accommodates sequences as bulky as Trp-Tyr-Tyr in different subsites (S 1 to S‘ 2, S 2 to S‘ 1, and probably S 3 to S 1 ). Several bitter peptides were identified in the digests.