豆粕血管紧张素转化酶抑制肽的结构鉴定及作用机制解析

Identification and Mechanism Analysis of Angiotensin-I Converting Enzyme Inhibitory Peptides from Soybean Meal

以大豆粕为原材料,利用超声辅助酶解技术、超滤-?KTA层析相结合的方法分离纯化获取豆粕酶解产物中血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制肽,对其分子质量分布进行研究,后通过质谱分析与分子对接技术鉴定并筛选出ACE抑制活性肽的氨基酸序列,经固相合成肽序列,检测其ACE抑制肽的活性并基于分子对接技术探索其抑制机制。结果表明:经超声辅助酶解提取获得的豆粕肽分子质量主要分布在6 000 Da以下;根据分子对接的最低预测自由能筛选出的GVRP(-8.44 kcal/mol)和IIVTP(-9.04 kcal/mol)可以抑制ACE活性,半抑制浓度(50%inhibitory concentration,IC_(50))分别为(84±0.06)、(77±0.08)μmol/L;分子对接结果表明:GVRP、IIVTP能够与ACE的活性口袋S1、S1′、S2形成氢键相互作用,共有的过近接触(3.5?范围内)ACE氨基酸残基为His513、Ala354和Glu384。本研究基于串联质谱与分子对接技术,建立从混合多肽中快速鉴定、筛选活性多肽的方法,探究活性多肽与ACE稳定结合并体现其ACE活性的抑制机制,为后续的深入研究提供一定参考。

In this study,ultrasonic-assisted enzymatic hydrolysis,ultrafiltration andÄKTA chromatography were sequentially used to separate and purify angiotensin-converting enzyme(ACE)inhibitory peptides from an enzymatic hydrolysate of soybean meal,and the molecular mass distribution of peptides was investigated.Then,the amino acid sequence of the ACE inhibitory peptides was identified by mass spectrometry(MS)and molecular docking.A consensus peptide sequence was synthesized by a solid-phase method to determine its ACE inhibitory activity and explore its underlying mechanism by molecular docking.The results showed that after ultrafiltration,the molecular mass distribution of peptides in the hydrolysate was mainly under 6000 Da.The peptides GVRP and IIVTP,selected for their lowest free energy(–8.44 and–9.04 kcal/mol correspongdingly)predicted by molecular docking,could inhibit ACE activity,with half-maximum inhibitory concentration(IC_(50))values of(84±0.06)and(77±0.08)μmol/L,respectively.The molecular docking results revealed that GVRP and IIVTP could form hydrogen bonds with the S1,S1′and S2 pockets of ACE.The shared amino acid residues in close contacts(within 3.5Å)with ACE were His513,Ala354 and Glu384.Based on the LC-MS/MS and the molecular docking technology,a method to rapidly identify and select active ACE inhibitory peptides from peptides mixture was established.The stable binding of active peptides to ACE and the inhibitory mechanism of its ACE activity were also reflected in this study.The finding of this study may provide further references for studies on ACE inhibitory peptides.