Preparation Of ACE-inhibitory Peptides From Bombyx Mori Silk Fibroin And Their Physicochemical Properties

Hypertension is a chronic disease that can induce cardiovascular and cerebrovascular diseases,which seriously endangers people's health.Some synthetic drugs such as captopril,enalapril,and fosinopril have been used to cure high blood pressure clinically,but these drugs always trigger taste disorders and dry cough.The angiotensin-converting enzyme?ACE?inhibitory peptides is one of the most effective natural drugs to treat hypertension,due to its high security,inhibitory effects,easy absorption and without side effects.Silk fibroin is widely distributed with high percentage hydrophobic amino acid in Bombyx mori.It is a potential protein resource for ACE inhibitory peptides preparation.To date,there were few studies on the molecular mechanism of silk fibroin ACE inhibitory peptides to cure blood pressure lowering.In the present study,silkworm silk fibroin was used to prepare ACE inhibitory peptide by using enzymatic hydrolysis method.Silk fibroin ACE inhibitory peptide was extracted and identified by using ultrafiltration,following ion exchange chromatography,dextran gel separation chromatography,RP-HPLC and LC/MS,respectively.The molecular mechanisms of the anti-high blood pressure of the silk fibroin ACE inhibitory peptide were investigated by using Molecular docking and Lineweaver-Burk double reciprocal mapping.Results showed that alcalase was the optimal hydrolase for the preparation of silk fibroin ACE inhibitory peptide;factors that affected the ACE inhibitory peptide activity were:substrate concentration>enzymatic dosage>enzymatic hydrolysis time>p H value.The optimum conditions for enzymatic hydrolysis were as follows:enzymatic hydrolysis temperature 45°C,enzymatic hydrolysis time 61.66 min,p H 8.54,substrate concentration4.95%,and enzyme dosage 3140.23 U/g.After ultrafiltration separation,the fraction MW<5 k Da had the strongest ACE inhibitory activity;UV spectroscopy indicated that the fraction could cause a structural change on ACE.In vitro digestion experiments showed that ACE inhibitory peptides from silk fibroin had good acidic,alkaline,and thermal stability and ability of anti-intestinal digestion.The effective constituent was obtained(IC₅₀value was 0.00625 mg/m L)from the small fractions by using DEAE-52 exchange chromatography,Sephadex G-50 gel chromatography,Sephadex G-15 gel separation chromatography and RP-HPLC.The LC/MS analysis showed that the molecular weight of the silk fibroin ACE inhibitory peptides ranged from 162.13 to 542.37 Da,which were mainly composed of 2?4 peptides.Amino acid sequences of five ACE inhibitory peptides were predicted.They were SG or GS,SV or DA,YT,EECY and PAL.The molecular docking studies revealed that the ACE inhibition of SG was mainly attributed to forming strong hydrogen bonds with the S1 pocket?Glu384,Ala354,Tyr523?and the S2 pocket?His353,His513,Lys511,Tyr520?.GS was mainly attributed to forming strong hydrogen bonds with the S1 pocket?Tyr523,Ala354,Glu384?.SV mainly interacted with the S1 pocket?Ala354?and S2 pocket?Tyr520,Gln281,His513,His353?.DA mainly interacted with the S1 pocket?Ala354?and S2 pocket?Gln281,His353?and near Zn²⁺?His383 and His387?.YT mainly interacted with ACE amino acid residues Glu123,Asn85,Tyr62,Ser517 and Arg124.PAL mainly interacted with ACE amino acid residues Tyr360 and Arg522.EECY mainly interacted with ACE amino acid residues Arg522,Glu123,Lys118 and Arg124.PAL had the smallest IC₅₀ value of 0.0018 mg/ml.The Lineweaver-Burk plots suggested that SG?k_i=3.033±0.512 mmol/L?and EECY?k_i=3.845±0.716 mmol/L?were competitive inhibitors against ACE.DA?k_i=9.600±0.602mmol/L?,SV?k_i=3.492±0.106 mmol/L?and GS?k_i=6.458±0.644 mmol/L?were non-competitive inhibitors against ACE.PAL?k_i=0.617±0.50 mmol/L?and YT?k_i=1.146±0.044 mmol/L?are anti-competitive inhibitors against ACE,among which PAL has the smallest k_i and the strongest affinity with ACE.