Subcritical Water Processing of Proteins: An Alternative to Enzymatic Digestion?

Subcritical water is an emerging tool in the processing of bioorganic waste. Subcritical 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 subcritical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of subcritical water with respect to the production of peptides from three model proteins, hemoglobin, bovine serum albumin, and beta-casein, and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of subcritical water (SCW) treatment on two protein post translational modifications, disulfide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following subcritical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild subcritical water conditions (160 degrees C), all proteins showed favored cleavage of the Asp-X bond. The results for beta-casein revealed favored cleavage of the Glu-X bond at subcritical water temperatures of 160 and 207 degrees C. That was similarly observed for bovine serum albumin at a subcritical water temperature of 207 degrees C. Subcritical water treatment results in very limited cleavage of disulfide bonds. Reduction and alkylation of proteins either prior to or post subcritical water treatment improve reported protein sequence coverages. The results for phosphoprotein beta-casein show that, under mild subcritical water conditions, phosphorylation may be retained on the peptide hydrolysis products.