Towards a Quantitative Description of Proteolysis: Contribution of Demasking and Hydrolysis Steps to Proteolysis Kinetics of Milk Proteins.

The hydrolysis of proteins by proteases (proteolysis) plays a significant role in biology and food science. Despite the importance of proteolysis, a universal quantitative model of this phenomenon has not yet been created. This review considers approaches to modeling proteolysis in a batch reactor that take into account differences in the hydrolysis of the individual peptide bonds, as well as the limited accessibility (masking) for the enzymes of some hydrolysis sites in the protein substrate.

Modeling of the Peptide Release during Proteolysis of β-Lactoglobulin by Trypsin with Consideration of Peptide Bond Demasking.

Prospects for predicting the fragmentation of polypeptide chains during their enzymatic hydrolysis using proteolysis models are considered. The opening of the protein substrate during proteolysis and the exposure of its internal peptide bonds for a successful enzymatic attack, the so-called demasking process, were taken into account. The two-step proteolysis model was used, including the parameters of demasking and the rate constants of hydrolysis of enzyme-specific peptide bonds.

Proteolysis of Micellar β-Casein by Trypsin: Secondary Structure Characterization and Kinetic Modeling at Different Enzyme Concentrations.

Tryptic proteolysis of protein micelles was studied using β-casein (β-CN) as an example. Hydrolysis of specific peptide bonds in β-CN leads to the degradation and rearrangement of the original micelles and the formation of new nanoparticles from their fragments. Samples of these nanoparticles dried on a mica surface were characterized by atomic force microscopy (AFM) when the proteolytic reaction had been stopped by tryptic inhibitor or by heating.

Modeling of Proteolysis of β-Lactoglobulin and β-Casein by Trypsin with Consideration of Secondary Masking of Intermediate Polypeptides.

The opening of protein substrates during degradation by proteases and the corresponding exposure of their internal peptide bonds for a successful enzymatic attack, the so-called demasking effect, was studied for β-lactoglobulin (β-LG) and β-casein (β-CN) hydrolyzed by trypsin. Demasking was estimated by monitoring the redshift in intrinsic tryptophan fluorescence, characterizing the accessibility of polypeptide chains to aqueous medium. The secondary masking of intermediate polypeptides, giving an inverse effect to demasking, caused a restriction of the substrate opening.

Kinetic modeling of demasking and hydrolysis of peptide bonds during proteolysis of β-lactoglobulin by trypsin.

Proteolysis of β-lactoglobulin by trypsin was studied with fluorescence spectroscopy and an empirical exponential model was engaged to describe the peptide bond hydrolysis kinetics. The shift in the fluorescence maximum of tryptophan residues, from 342 to 352 nm, in the course of β-lactoglobulin degradation was used as an indicator of the transition of masked peptide bonds to the demasked ones, which were accessible for the enzyme action. A simple equation with only two parameters was suggested to link together the degree of demasking of peptide bonds and the degree of their hydrolysis, allowing the kinetic description of proteolysis.