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 beta-casein (beta-CN) as an example. Hydrolysis of specific peptide bonds in beta-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. The changes in the content of beta-sheets, alpha-helices, and hydrolysis products during proteolysis were estimated by using Fourier-transform infrared (FTIR) spectroscopy. In the current study, a simple kinetic model with three successive stages is proposed to predict the rearrangement of nanoparticles and the formation of proteolysis products, as well as changes in the secondary structure during proteolysis at various enzyme concentrations. The model determines for which steps the rate constants are proportional to the enzyme concentration, and in which intermediate nano-components the protein secondary structure is retained and in which it is reduced. The model predictions were in agreement with the FTIR results for tryptic hydrolysis of beta-CN at different concentrations of the enzyme.

Automated summary

In this study, the tryptic proteolysis of protein micelles was investigated using beta-casein as a model. The hydrolysis of specific peptide bonds in beta-casein resulted in the degradation and rearrangement of the original micelles, leading to the formation of new nanoparticles. The changes in the secondary structure and hydrolysis products during proteolysis were examined using Fourier-transform infrared spectroscopy. A simple kinetic model with three successive stages was proposed to predict the rearrangement of nanoparticles and the formation of proteolysis products, as well as changes in the secondary structure at different enzyme concentrations. The model predictions agreed well with the experimental results.