Study Of Angiotensin-converting Enzyme Inhibitory Peptides Based On Protease-specific Hydrolysis

Angiotensin-converting enzyme(ACE)inhibitors are now being used as an effective treatment for hypertension.Although commercially available chemically synthesized drugs are effective in the treatment of hypertension,these drugs often cause side effects such as cough,angioedema,and hypotension.Safer food-derived ACE-inhibiting peptides are considered to be a safer and more effective treatment for this chronic disease.Small peptides of tetrapeptide and below have gained a lot of attention from a wide range of researchers due to their easy absorption by the body.Most of the foodderived ACE inhibitory peptides found so far were obtained by traditional experimental screening,which is time-consuming and blind.Based on the previous research on 8000 tripeptides and 160,000 tetrapeptides,it was concluded that tryptophan is the characteristic amino acid in the oligopeptide ACE inhibitory peptides.In this work,a rabbit-derived skeletal myosin light chain C of natural origin without tryptophan was screened.Based on this,its tryptophan-containing mutants were obtained by E.coli expression system,and the ACE inhibitory effect of the tryptophan-containing peptide was verified by chymotrypsin hydrolysis assay.The ACE inhibition rate of the hydrolysis product of mutant 2 with more tryptophan was 54.54%,which was significantly higher than that of the wild type with 40.91% inhibition rate.In addition,the recombinant proteinase K,which can specifically hydrolyze aromatic and hydrophobic amino acids,was successfully expressed by the Pichia pastoris expression system.The ACE inhibition rate of the hydrolysis product further increased after the combined hydrolysis of chymotrypsin and proteinase K.The ACE inhibition rate of the hydrolysis product of the tryptophan-containing mutant 2 was increased by 57.61% compared with that of chymotrypsin alone,which was more obvious than that of the tryptophan-free wild type hydrolysates.Mass spectrometry detected the presence of terminal tryptophan-containing peptides in the hydrolysis products,and molecular docking and molecular dynamics simulations further explained the mechanism of ACE inhibition by these peptides.The hydrolysis of specific proteins in vitro carried out in this project verified the important role of tryptophan in ACE inhibitory peptides and confirmed the feasibility of obtaining ACE inhibitory peptides by protease-specific hydrolysis,which provided a theoretical basis for the development of new ACE inhibitory peptides.