CN114736268A - 一种提高活性肽ace抑制率的修饰方法、ace抑制肽及其应用 - Google Patents
一种提高活性肽ace抑制率的修饰方法、ace抑制肽及其应用 Download PDFInfo
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Abstract
一种提高活性肽ACE抑制率的修饰方法、高ACE抑制率的ACE抑制肽及其应用,属于肽修饰技术领域。本发明对YLVR活性肽进行酶法修饰,修饰后的混合物在0.25mg/mL浓度下的ACE抑制率由52.58%提高至93.56%。根据NANO‑HPLC‑MS/MS鉴定结果,首次发现肽链的N端发生氨基酸聚类会提高肽ACE抑制率,基于此发现,本发明设计了数据库中未被鉴定出的3种高ACE抑制率的ACE抑制肽YLLVR、YYLVR和YYLLVR,经此方法得到的肽活性较高,可作为潜在的降压成分,可以用于制备ACE抑制剂、血管紧张素转换酶抑制剂、降血压药物或具有降血压的功能食品,对资源的综合利用具有极大意义。
Description
技术领域
本发明属于肽修饰技术领域,具体涉及一种提高活性肽ACE抑制率的修饰方法、高ACE抑制率的ACE抑制肽及其应用。
背景技术
高血压为当今世界最常见的心血管疾病之一,是冠心病、脑卒中最常见的危险因素。据报道,我国18岁及以上成人高血压患病率为23.0%,患病人数达2.435亿,正常高值血压患病率为41.4%,患病人数4.363亿。高血压知晓率、治疗率、控制率及治疗控制率分别为42.7%、38.3%、14.5%和38.0%,且患病率随年龄增高而上升。每年由其诱发的心脑血管疾病死亡人数占总死亡人数的51%。因此,控制血压被认为是提高全球健康,降低心脑血管疾病的关键目标。
血管紧张素转换酶(angiotensin converting enzyme,ACE)是介导机体引发高血压的关键酶促因子,ACE抑制肽能够抑制ACE活性。来自食物的ACE抑制肽被认为是一种无毒、易在人体内代谢等特点的天然物质,这些肽来源于食物蛋白质,没有降压药物带来的一系列副作用,并且越来越多的食源肽被证明具有降压功效。但是,酶解得到的活性肽是原料蛋白中存在的,它的活性因此受到制约,通过酶法水解无法进一步提高活性肽的活性。对已有的ACE抑制肽进行修饰也是一种行之有效的方法。已有研究表明添加蛋白酶对肽分子进行分子改造可以减少肽的苦味并提高其ACE抑制率,但具体结构变化机理未知。
发明人在前期研究中,以榛仁及松仁分离蛋白为原料,制备、纯化并利用LC-MS/MS结合De novo结构解析得到多个序列明确的高活性ACE抑制肽段(Exploration of themolecular interactions between angiotensin-I-converting enzyme(ACE)and theinhibitory peptides derived from hazelnut(Corylus heterophylla Fisch.),Chunlei Liu,Wei-hong Min)。故本研究尝试以鉴定得到,并通过大鼠实验证明具有高ACE抑制率的ACE抑制肽YLVR为原料,利用修饰反应以提高其ACE抑制活性。另外,采用NANO-HPLC-MS/MS鉴定方法对修饰后的肽段进行结构鉴定,分析酶法修饰反应中肽分子重组机制,本发明将为酶法修饰肽技术的深入研究和ACE抑制肽的设计提供理论依据,对资源的综合利用具有极大的意义。
发明内容
本发明的目的之一是提供一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:称取一定质量的活性肽,加入蒸馏水,使活性肽质量浓度为10%~40%,再用0.5mol/L的NaOH溶液调节反应溶液的pH值为6~10;然后加入活性肽质量0.1%~10%的蛋白酶,于20~55℃水浴中反应3.5~7.5h,反应结束后迅速于85~95℃加热10~20min使酶灭活,再立即冷却至室温;最后,冷冻(-50~-40℃)干燥得到高ACE抑制率的ACE抑制肽,4℃保存。
活性肽的来源为化学固相合成、动物源、植物源或微生物源中的一种,特别的为YLVR(化学固相合成,Exploration of the molecular interactions betweenangiotensin-I-converting enzyme(ACE)and the inhibitory peptides derived fromhazelnut(Corylus heterophylla Fisch),Food Chemistry 245(2018)471-480)。
蛋白酶为胃蛋白酶、碱性蛋白酶、中性酶、木瓜蛋白酶或胰蛋白酶中的一种,特别的为碱性蛋白酶Alcalase 2.4L。
本发明的目的之二是分析酶法修饰反应中肽分子重组机制及修饰机理,提高3种高ACE抑制率的ACE抑制肽。目前对修饰后肽序列的改变缺少鉴定,以及对这种结构的改变与ACE抑制活性改变之间联系的研究也较少。本发明对修饰后的ACE抑制肽进行序列鉴定,采用NANO-HPLC-MS/MS技术并经从头测序方法进行质谱解析,对鉴定结果进行统计学研究,设计出3种高ACE抑制率的ACE抑制肽YLLVR(Tyr-Leu-Leu-Val-Arg)、YYLVR(Tyr-Tyr-Leu-Val-Arg)和YYLLVR(Tyr-Tyr-Leu-Leu-Val-Arg),其结构式如下所示。本发明旨在为降血压药物合成和ACE抑制肽构效关系研究提供理论基础。
本发明的目的之三是应用本发明所述的ACE抑制肽YLLVR、YYLVR和YYLLVR制备ACE抑制剂、血管紧张素转换酶抑制剂、降血压药物或具有降血压的功能食品,具体是以抑制肽YLLVR、YYLVR、YYLLVR中的一种或两种为活性成分,添加药物或食品中可接受的载体或辅料制备得到。
本发明相比现有技术的有益效果为:
本发明公开了一种ACE抑制肽的酶法修饰方法,此方法会显著提高活性肽ACE抑制率,其次通过修饰反应可以掩盖榛仁肽(YLVR)的苦味。修饰后的混合物在0.25mg/mL浓度下的ACE抑制率由52.58%显著提高至93.56%。本发明公布的修饰方法路线设计简单合理,反应条件温和且环保,无不良副产物生成,且修饰后产物的ACE抑制率显著提高,使活性肽可以更好的作为天然ACE抑制剂添加到食品或药品中。
通过质谱数据深入挖掘酶法修饰活性肽的机理,首次发现相同氨基酸在肽链N端聚类会提高活性ACE抑制率,在质谱解析中,针对NCBI中已鉴定的植物蛋白数量较少的情况,采用常规的Uniprot全序列数据库搜索与分析并不适用,因此采用de novo法确定组分的序列结构可以防止活性组分的遗漏。基于对鉴定结果解析,首次发现肽链的N端发生氨基酸聚类会提高肽ACE抑制率,鉴别与设计得到3种数据库中未被鉴定出的ACE抑制肽,在0.25mg/mL浓度下,YLLVR的ACE抑制率为72.61%,YYLVR的ACE抑制率为66.35%,YYLLVR的ACE抑制率为89.10%。修饰后低苦味的高ACE抑制活性肽作为保健品以及食品的原料,应用于食品与医药行业中,可用于相关疾病预防。
附图说明
下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1得到的修饰肽的ACE抑制率图;
图2为本发明所述的3种ACE抑制肽的质谱图;其中图(a)对应YLLVR,图(b)对应YYLVR,图(c)对应YYLLVR。
ACE抑制率的测定:
将实施例1活性肽(YLVR)、实施例1步骤(1)产物(即修饰后得到的混合物)、YLLVR、YYLVR和YYLLVR溶液(浓度均为0.25mg/mL)和ACE溶液(0.25mU/mL,50μL)在37℃下水浴10min,然后加入150μL、4.15mM HHL(马尿酸)底物在37℃下水浴反应1h,加入250μL、1M HCl终止反应。加入500μL乙酸乙酯震荡提取生成的马尿酸,以3000r/min条件离心10min,将200μL上清液置于110℃的干燥烘箱中直至溶液完全蒸发。将残余物溶解在1mL蒸馏水中,并使用酶标仪在228nm下测量其UV光谱密度。
如图1所示,其中混合物对应实施例1步骤(2)产物,修饰后的肽ACE显著提高,表明本发明提供的修饰方法可以显著提高ACE抑制率;NANO-HPLC-MS/MS鉴定得到的YLLVR、YYLVR和YYLLVR有ACE较强的抑制活性。
如图2所示,表明得到了YLLVR、YYLVR和YYLLVR目标结构产物。
具体实施方式
以下结合实施例来进一步解释本发明,但实施例并不对本发明作任何限定。
实施例1:
(1)肽的修饰
取2g冷冻干燥后的活性肽(YLVR),用蒸馏水配成活性肽质量浓度为36%的溶液,用0.5mol/L NaOH调pH值8.3,然后加入肽质量1.50%的碱性蛋白酶(Alcalase 2.4L),于40.8℃水浴中反应5h,反应结束后迅速于90℃下加热15min使酶灭活;-45℃冷冻干燥24h,4℃保存。
(2)质谱鉴定
将样品经由配备在线纳喷离子源的HPLC-MS/MS分析。整套系统为串联EASY-nanoLC 1200的Q ExactiveTM质谱仪。共上样3μL样品(分析柱:Acclaim PepMap C18,75μm x25cm),以60min的梯度分离样品,柱流量控制在300nL/min,柱温为40℃,电喷雾电压2kV,梯度从2%的B相起始,平衡3min,在47min以非线性梯度升高到35%,1min内升高到100%,维持12min。
质谱仪在数据依赖采集模式下运行,自动在MS和MS/MS采集间切换。质谱参数设置如下:(1)MS:扫描范围(m/z):200-1500;分辨率:70,000;AGC target:3e6;最大注入时间:60ms;(2)HCD-MS/MS:分辨率:17,500;AGC target:5e4;最大注入时间;50ms碰撞能量:27;动态排除时间:20s。
串联质谱图经过PEAKS Studio version 10.6(Bioinformatics SolutionsInc.,Waterloo,Canada)分析。PEAKS DB对uniprot-Betulaceae(version202110,36905entries)数据库搜库,设置none酶解。搜库参数碎片离子质量容许误差:0.02Da,母离子质量容许误差:7ppm,可变修饰:Oxidation(M)15.99,Deamidation(NQ)0.98,Acetylation(Protein N-term)42.01。蛋白卡值为:-10lgP≥0,至少含1unique peptide;肽段卡值为:-10lgP≥15。
以上所述仅为本发明的较佳实施方式,但本发明的保护范围并不局限于此。凡依本发明申请专利范围所做的均等改变与修饰,都应包含在本发明的保护范围之内。
Claims (8)
1.一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:称取一定质量的活性肽,加入蒸馏水,使活性肽质量浓度为10%~40%,再用0.5mol/L的NaOH溶液调节反应溶液的pH值为6~10;然后加入活性肽质量0.1%~10%的蛋白酶,于20~55℃水浴中反应3.5~7.5h,反应结束后迅速于85~95℃加热10~20min使酶灭活,再立即冷却至室温;最后,冷冻干燥得到高ACE抑制率的ACE抑制肽。
2.如权利要求1所述的一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:活性肽为化学固相合成、动物源、植物源或微生物源中的一种。
3.如权利要求2所述的一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:其特征在于:活性肽为YLVR。
4.如权利要求1所述的一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:蛋白酶为胃蛋白酶、碱性蛋白酶、中性酶、木瓜蛋白酶或胰蛋白酶中的一种。
5.如权利要求4所述的一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:蛋白酶为碱性蛋白酶Alcalase 2.4L。
6.如权利要求1所述的一种提高ACE抑制肽ACE抑制率的修饰方法,其特征在于:冷冻干燥的温度为-50~-40℃。
8.权利要求7所述的高ACE抑制率的ACE抑制肽在制备ACE抑制剂、血管紧张素转换酶抑制剂、降血压药物或具有降血压功能食品中的应用。
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