SummaryThe proteolytic specificity of chymosin (EC 3.4.23.4) on bovine αs1-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(?), Phe150–Phe153, Phe150–Leu156, Tyr154–Tyr159, Tyr154–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 Arg1-Phe23 and Tyr165–Trp199 were the first and second peptides produced under both conditions. Pathways are proposed to interpret the proteolysis of αs1-casein in solution under the conditions of this study.

Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry. Most of the peptides were from the N-terminal half of β-casein, but peptides from αs1- and αs2-caseins were also identified; the extract also contained α-lactalbumin. Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from αs1- and β- caseins, produced by chymosin and plasmin respectively. Plasmin seemed to be involved in the hydrolysis of αs2-casein. Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.

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SUMMARYThe water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes. Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl, pH 8·6, and reversed-phase HPLC or electroblotting from urea-PAGE gels. Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry. Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from β-casein, especially from a short region in the N-terminal half of the molecule. Only six peptides originated from αs1-casein; β-lactoglobulin was also identified in the retentate. The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.

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