摘要
为优化酶解法制备绵羊乳酪蛋白ACE抑制肽的工艺条件以及筛选和鉴定一种新的ACE抑制肽,选用5种蛋白酶水解酪蛋白,以水解度、分子质量分布和ACE抑制率为指标筛选最适蛋白酶,采用单因素和响应面试验优化工艺,采用三合一质谱仪(Orbitrap Fusion Lumos Tribrid MS)方法鉴定分子质量小于3 ku组分的氨基酸序列,筛选潜在ACE抑制肽,进行人工合成,测定其IC50值。采用Linewaver-Burk作图确定酶抑制动力学,结合分子对接解析肽段的抑制机制。结果表明:碱性蛋白酶水解酪蛋白的最佳条件为pH 6、底物含量8%、酶添加量4%、温度55℃、水解时间90 min,此时酪蛋白水解液ACE抑制率为99.1%。验证具有ACE抑制活性的肽段10条,筛选出一条新颖的降血压肽——LFRQFY(源自αs1-酪蛋白),其ACE抑制活性的IC50为(7.9±1.7)μmol/L,酶抑制动力学为混合抑制模式。分子对接结果表明:LFRQFY能与ACE的氨基酸残基Ala354(活性口袋S1)、His353(活性口袋S2)形成氢键,具有显著的体外降血压活性。
In order to optimize the technological conditions for preparing ACE inhibitory peptide of sheep casein by enzymatic hydrolysis,and a new ACE inhibitory peptide was screened and identified from casein hydrolysate.ACE inhibitory peptide was prepared from sheep cheese protein by five proteases enzymatic hydrolysis,and taking molecular weight distribution,degree of hydrolysis and inhibition rate of angiotensin-I-converting enzyme(ACE)as indicators to screening the most suitable protease.The single factor and response tests were used to optimize enzymatic hydrolysis conditions.The amino acid sequences of components less than 3 ku were identified by Orbitrap Fusion Lumos Tribrid MS,and the potential ACE inhibitory peptides were screened and determining their IC50 values after synthesis.The inhibition kinetics of enzyme was determined by Linewaver-Burk mapping,and molecular docking was used to explain the mechanism of peptides inhibition of ACE.The results showed that alkaline protease was the most suitable protease,the optimal enzymolysis conditions were as follows:pH 6,substrate concentration of 8%,enzyme/substrate ratio(E/S)of 4%,temperature at 55℃and enzymatic hydrolysis time for 90 min.The ACE inhibition rate of casein antihypertensive peptide prepared under this condition was 99.1%.A total of 484 peptides were identified in the fraction less than 3 ku,among which 10 peptides had been verified to have ACE inhibitory activity.A novel peptide LFRQFY(derived fromαs1-casein)were found to have highly inhibition potency with IC50 values of(7.9±1.7)μmol/L and exhibiting mixed inhibition patterns.The molecular docking results revealed that LFRQFY were mainly attributed to forming very strong hydrogen bonds with ACE amino acid residues Ala354(S1 pocket)and His353(S2 pocket),and showed significant antihypertensive activity in vitro.
作者
汤海霞
张艳
葛武鹏
宋宇轩
王海燕
王爽爽
Tang Haixia;Zhang Yan;Ge Wupeng;Song Yuxuan;Wang Haiyan;Wang Shuangshuang(College of Food Science and Engineering,Northwest A&F University,Yangling 712100,Shaanxi;Fuping County Inspection and Testing Center,Shaanxi Goat Milk Product Quality Supervision and Inspection Center,Fuping 711700,Shaanxi;College of Animal Science and Technology,Northwest A&F University,Yangling 712100,Shaanxi)
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2022年第6期220-231,共12页
Journal of Chinese Institute Of Food Science and Technology
基金
陕西省重点研发计划项目(2021ZDLNY02-09)。
关键词
ACE抑制肽
绵羊乳
分子质量分布
分子对接
响应面
ACE inhibitory peptide
sheep milk
the molecular weight distribution
molecular docking
response surface methodology
