|Title||Towards predicting enzymatic protein hydrolysis|
|Source||Wageningen University. Promotor(en): H. Gruppen, co-promotor(en): P.A. Wierenga; H.A. Schols. - Wageningen : Wageningen University - ISBN 9789463432351 - 190|
|Publication type||Dissertation, internally prepared|
The experimental maximum degree of hydrolysis (DHmax,exp) was typically lower than the theoretical maximum degree of hydrolysis (DHmax,theo), calculated using the number of cleavage sites (as based on protease specificity) on the protein. The aim of this thesis is, therefore, to develop a method to better define expectations of the DHmax,exp. The developed method is based on the molecular properties of the protein and the protease, and the effects of modifications on protein primary structure and changes in system conditions. Protein primary structure was modified by glycation, the initial stage of the Maillard reaction upon heating proteins in the presence of carbohydrates. The effect of glycation on DHmax,exp for five different enzymes could be predicted based on the protease primary and secondary specificity, and the sensitivity of the enzyme to modifications of the binding site. Secondary specificity was determined by linking the molecular properties of the amino acids on the binding site positions of cleavage sites with the enzyme selectivity. The enzyme selectivity, i.e. the relative hydrolysis rate constants towards cleavage sites in a protein, of bovine, porcine and human trypsins was determined after full quantitative analysis of peptides formed during hydrolysis. The prediction of DHmax,exp for bovine trypsin based on the secondary specificity was 5 times better than the prediction based on the primary specificity (DHmax,theo). The large differences found in DHmax,exp by the three trypsins could be predicted based on enzyme secondary specificity. The substrate concentration, one of the system conditions varying in the in vitro digestion models, also showed large effect on DHmax,exp. This was found to be related to changes in the mechanism of hydrolysis, illustrated by the differences in the percentage of intact protein in the hydrolysates as a function of DH. Due to the changes in mechanism, different amounts of inhibitory peptides for the protease were proposed to be present in the hydrolysates, leading to various DHmax,exp. Based on these findings, a model to predict the DHmax,exp of hydrolysis of any given protein by any specific protease was developed. The concept was shown to adequately explain the hydrolysis of proteins with various structural states and in hydrolysis by a sequential addition of enzyme.