Valorization of cruor slaughterhouse by-product by enzymatic hydrolysis for the production of antibacterial peptides: focus on α 1-32 family peptides mechanism and kinetics modeling.

Bovine hemoglobin is the major component of the cruor (slaughterhouse by-product) and can be considered as an important source of active peptides that could be obtained by pepsic hydrolysis. The kinetics of appearance and disappearance of several antibacterial peptides from α 1-32 family during hydrolysis of synthesized α 1-32 peptide, of purified bovine hemoglobin and of cruor was studied, and reaction scheme for the hydrolysis of α 1-32 family peptides from these three sources was determined. On this basis, a mathematical model was proposed to predict the concentration of each peptide of interest of this family depending on hydrolysis time, and also on temperature (in the range 15-37 °C), pH (in the range 3.5-5.5) and enzyme to substrate ratio (in the range 1/50-1/200 for the synthesized peptide and 1/5-1/20 for purified bovine hemoglobin and cruor). Apparent rate constants of reactions were determined by applying the model on a set of experimental data and it was shown that they depended on the temperature according to Arrhenius's law, that their dependence on the pH was linear, and that enzyme to substrate ratio influence was limited (in the studied range).

Mechanism and kinetics modeling of the enzymatic hydrolysis of α1-32 antibacterial peptide.

Several antibacterial peptides can be obtained by enzymatic hydrolysis of the α chain of bovine hemoglobin. The kinetics of α1-32 peptide hydrolysis by pepsin was studied at several temperatures (15, 23 and 37 °C). Intermediate and final peptides were identified, and their antibacterial activity was assessed against four bacterial species. Evolution of generated peptides concentration enabled to propose a reaction pathway describing the parallel and consecutive reactions taking place during the hydrolysis. A mathematical model, based on the proposed mechanism, was developed to describe the kinetics of generated peptides during α1-32 hydrolysis. The constants of the main reactions were identified based on the experimental data, and their dependence on temperature was established using Arrhenius-type equations. Validation of the proposed model was performed by predicting kinetics of α1-32 peptide hydrolysis at 30 °C (all other experimental conditions being unchanged) with a good accuracy. This mathematical model could allow defining the optimal conditions for the production of various intermediate peptides with antibacterial activity from peptic hydrolysis of α1-32 peptide.