Characterization of casein phosphopeptides

© 1995 Dr. Nicholas J. AdamsonCasein phosphopeptides (CPP), containing the cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, have been shown to stabilize amorphous calcium phosphate at neutral and alkaline pH and to be anticariogenic in various in vitro, animal and human experiments. Anticariogenic casein phosphopeptides (ACPP) therefore have commercial potential as toothpaste, mouthwash and food additives for the prevention of dental caries. The aim of this project was to comprehensively characterize the CPP produced under industrially-relevant conditions using the commercially available enzymes Novo trypsin PTN 3.0 S, Novo Alcalase® 2.4L and Pancreatin 4NF. To facilitate studies on the identification and characterization of CPP a simple and efficient purification procedure involving selective precipitation of Ca2+/ethanol-induced aggregates of the phosphopeptides from enzymic digests was developed. The individual peptides of the precipitates were purified using anion exchange FPLC and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapour-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalysed β-elimination in the presence of ethanethiol. Alternatively, in the event of low coupling efficiency to arylamine membranes, phosphoseryl residues were converted to S-ethylcysteinyl or S-propylcysteinyl residues and sequenced from polybrene-treated glass fibre discs. A method for the separation and identification of CPP using high performance capillary electrophoresis (HPCE) was also developed. The ability of HPCE to rapidly resolve phosphopeptides exhibiting varying degrees of phosphorylation, truncation and deamidation make it ideal for the product quality control of CPP. HPCE was also used to develop relationships between absolute electrophoretic mobility (µ) and peptide charge and size for CPP containing 2-5 phosphoseryl residues, with µ found to be proportional to q/M2/3 where q is the net negative charge and M is molecular mass of the peptide. The results showed that CPP can be produced using Novo trypsin and pancreatin with only minor modifications, relative to CPP produced using analytical-grade trypsin, such as slight truncation, deamidation and methionine oxidation. Deamidation and methionine oxidation most likely resulted from an elevated hydrolysis temperature and/or conditions employed during commercial casein (CN) production. Studies on the hydrolysis of casein at different enzyme:substrate (E/S) ratios by these enzymes suggest that for CPP production using Novo trypsin, a minimum degree of hydrolysis (DH) of 17.3% is required for maximal release of ACPP whilst for pancreatin, a DH of 19.0% is required. For pancreatin, it is recommended that this value is not exceeded so as to minimize the release of the truncated β-CN-4P(f7-24). CPP produced using alcalase were severely truncated relative to those prepared using Novo trypsin and pancreatin, resulting in the loss of residues suggested to be necessary for full anticariogenic activity. Decreasing E/S ratio slightly lowered the degree of truncation caused by alcalase, however, low E/S ratios also resulted in a reduction in CPP yield. It is likely that ACPP produced using alcalase would have much lower specific anticariogenic activity than those produced using Novo trypsin or pancreatin. In conclusion, it has been demonstrated that Novo trypsin PTN 3.0 S and Pancreatin 4NF are suitable enzymes for the production of CPP on a commercial scale and that Alcalase® 2.4L is unsuitable. For the production of CPP using Novo trypsin, a minimum DH value of 17.3% is required for maximal ACPP release whilst for pancreatin, a DH value of 19.0% is required. HPCE has been demonstrated to be an ideal method to monitor CPP quality