Sub-critical water processing of proteins: An alternative to enzymatic digestion?

Sub-critical water is an emerging tool in the processing of bioorganic waste. Sub-critical water is an environmentally benign solvent which has the potential to provide an alternative to traditional methods of protein hydrolysis without the inclusion of expensive acids or enzymes. To date, most studies on the sub-critical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of sub-critical water with respect to the production of peptides from three model proteins - haemoglobin, bovine serum albumin and β-casein - and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of SCW treatment on two protein post-translational modifications, disulphide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following sub-critical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild sub-critical water conditions (160 oC), all proteins showed favoured cleavage of the Asp-X bond. The results for β-casein revealed favoured cleavage of the Glu-X bond at sub-critical water temperatures of 160 oC and 207 oC. That was similarly observed for bovine serum albumin at sub-critical water temperature 207 oC. Sub-critical water treatment results in very limited cleavage of disulphide bonds. Reduction and alkylation of proteins either prior to or post sub-critical water treatment improves reported protein sequence coverages. The results for phosphoprotein β-casein show that under mild sub-critical water conditions, phosphorylation may be retained on the peptide hydrolysis products.