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

Abstract

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.

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

“…These ratios were chosen taking into account that in the previous study of hydrolysis of pure synthetic a 1-32 peptide, the ratios were 1/50, 1/100 and 1/200 [17], and that in hemoglobin, there was about ten times more peptide bonds to be hydrolyzed by pepsin than in the pure synthetic a 1-32 peptide [11,14,18].…”

“…Peptides of a 1-32 family (a 1-32, a 1-29, a 1-28, a 1-27 and a 1-23) generated during pepsic hydrolysis of synthetic a 1-32, hemoglobin and cruor, were identified and quantified using RP-HPLC and LC-ESI-MS analysis as described by Hedhili et al [17].…”

“…100 lL samples were taken at different hydrolysis times (t = 0, 1, 2.5, 5, 10, 15, 30 min, 1, 2, 3, 10, 24, 28 and 32 h) and the reaction was stopped by addition of 40 lL of disodium tetra borate (0.32 M, pH 12.7) to reach a pH of 10, as described previously [17].…”

“…In fact, a 1-32, a 1-29, a 1-28, a 1-27 and a 1-23 peptides are nontoxic and antibacterial peptides could be used for food, nutraceutical or pharmaceutical applications [14]. Pepsic hydrolysis of a 1-32 synthetic peptide was studied in a previous work [17].…”

“…Concentration evolution of the generated peptides was studied at different temperatures, leading to the establishment of the reaction pathway describing the consecutive and parallel reactions taking place during hydrolysis, and to the development of a model for kinetics of peptides generated during hydrolysis of a 1-32 synthetic peptide [17]. The objective of the present work was to generalize this model by applying it to the antibacterial peptides of a 1-32 family derived from the hydrolysis of real substrates such as bovine hemoglobin and cruor, and expand the field of application of this model integrating the effect of temperature, pH, and enzyme to substrate ratio (E/S) on the kinetics of hydrolysis.…”