Towards predicting protein hydrolysis by bovine trypsin

[Display omitted] •Bovine tryptic protein hydrolysis only reaches ∼48% of the theoretical maximum.•Limited extent of hydrolysis was caused by enzyme selectivity towards cleavage site.•Bovine trypsin selectivity was determined based on the peptide release kinetics.•Bovine trypsin prefers uncharged amino acids at P2 & P2′ of binding site.•Extent of hydrolysis and hydrolysate composition can be predicted by selectivity. The extent of protein enzymatic hydrolysis is considered to be mostly determined by protease specificity and the number of cleavage sites (CS) on the substrate. However, this theoretical maximum is typically not reached. The limited hydrolysis of certain CS may be due to the differences in enzyme preference resulting from neighbouring amino acids (AA) of the CS (secondary specificity). This study aims to find the link between enzyme secondary specificity and the relative hydrolysis rate constants (selectivity) of individual CS in a protein, to better predict the maximum experimental degree of hydrolysis. Bovine tryptic hydrolysis of α-lactalbumin and β-casein showed that ≥50% of the CS were inefficiently hydrolysed. The selectivity depended on the number of charged AA at P2 and P2′ positions of a CS. Trypsin efficiently cleaves CS with neutral AA at these two positions. The selectivity towards 12 out of 18 (67%) CS in β-lactoglobulin was correctly predicted. The predicted maximum degree of hydrolysis is within ∼13% error of the experimental value, which is ∼5 times better than the prediction based only on enzyme specificity. This work helps to estimate the extent of hydrolysis and the peptide formation of bovine tryptic hydrolysis of other substrates.