ENGINEERING OF TRYPSIN AND ITS IMPACT ON BETA-CASEIN PROCESSING

Tryptic processing of beta-casein yields several important nutraceutic and nutritious peptides. However, a final product peptide (1-105) sto ps the processing, inhibiting the enzyme. In attempt to modulate catal ytic properties of this protease, K188 was replaced with aromatic amin o acid residues. This aimed amplification of local hydrophobic and ele ctrostatic interactions at the substrate binding site. The catalytic p roperties of obtained mutants (K188F, K188Y, and K188W) were measured at pH 7, 8, 9, and 10 with synthetic substrates and beta-casein. Kinet ic analysis revealed that all the mutants conserve the capacity to spl it peptide bonds involving arginyl and lysyl residues. However, depend ing on mutation, the optimum pH of activity changes. As shown only by proteolysis of a natural substrate, produced mutants cleaved near 30 n ew peptide bonds compared to wild-type trypsin, 8 of them involving as paragine and glutamine amino acids. Some of the new cleavage sites can be related to the nature of the amino acid residue introduced in posi tion 188. Consequently, only the joint use of several methods (synthet ic substrate, protein substrate, influence of pH) can help to define b etter the differences of catalytic properties of wild-type and mutant proteases. Modifications introduced by the mutations are at the origin of the alteration of the specificity of the studied enzymes which are cleaving beta-casein in many places, hydrolysing well the fragment 1- 105. Since many tryptic inhibitors contain amidated Glu and Asp, and f orm amyloid structures, the new mutants could be used in hydrolysing r esistant proteic structures.