Comparison of the influence of pH on the selectivity of free and immobilized trypsin for β-lactoglobulin hydrolysis.

Although immobilized trypsin is a viable alternative to the free one in solution for producing protein hydrolysates, the change of selectivity introduced by immobilization is unclear. In this study, we compared the selectivity of free and immobilized trypsin towards different cleavage sites of β-lactoglobulin (β-Lg) with a focus on the impact of environmental pH. Both free and immobilized trypsin exhibited greater accessibility to native β-Lg at elevated pH (from pH 7.2 to 8.7). Additionally, free trypsin preferred to attack cleavage sites located at the C-terminus at pH 7.8, whereas an opposite preference for the N-terminus was observed at pH 8.7. Regarding the immobilized trypsin, the pH did not significantly influence its preference for the C- or N-terminus. Generally, immobilization of trypsin resulted in more focused cleavage within its specificity during the initial stage of hydrolysis, and some peptides were formed more rapidly by the immobilized trypsin.