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) stops the processing, inhibiting the enzyme. In attempt to modulate catalytic properties of this protease, K188 was replaced with aromatic amino acid residues. This aimed amplification of local hydrophobic and electrostatic interactions at the substrate binding site. The catalytic properties of obtained mutants (K188F, K188Y, and K188W) were measured at pH 7, 8, 9, and 10 with synthetic substrates and beta-casein. Kinetic analysis revealed that all the mutants conserve the capacity to split peptide bonds involving arginyl and lysyl residues. However, depending on mutation, the optimum pH of activity changes. As shown only by proteolysis of a natural substrate, produced mutants cleaved near 30 new peptide bonds compared to wild-type trypsin, 8 of them involving asparagine and glutamine amino acids. Some of the new cleavage sites can be related to the nature of the amino acid residue introduced in position 188. Consequently, only the joint use of several methods (synthetic substrate, protein substrate, influence of pH) can help to define better 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 form amyloid structures, the new mutants could be used in hydrolysing resistant proteic structures.
Chobert
J M
JM
Institut National de la Recherche Agronomique, Laboratoire d'Etude des Interactions des Molécules Alimentaires, Nantes, France.
Briand
L
L
Léonil
J
J
Mollé
D
D
Haertlé
T
T
D000595:Amino Acid Sequence; D002364:Caseins / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000235:genetics; D014357:Trypsin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D002364:Caseins; D011994:Recombinant Proteins; D014357:Trypsin