CN112442110B - 一种枸杞ace抑制肽和衍生多肽及应用、混合物 - Google Patents
一种枸杞ace抑制肽和衍生多肽及应用、混合物 Download PDFInfo
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Abstract
本发明涉及一种利用枸杞蛋白通过发酵法制备血管紧张素转换酶(angiotensin‑converting enzyme,ACE)抑制活性的多肽。多肽LEPIGVVGHI,其氨基酸序列为Leu‑Glu‑Pro‑Ile‑Gly‑Val‑Val‑Gly‑His‑Ile,分子量为1033.24Da。其具有良好的ACE抑制活性,具有开发成为调节血压的功能性食品或者是药物的良好应用前景。
Description
技术领域
本发明涉及一种通过发酵法将枸杞蛋白质制备具有血管紧张素转换酶(angiotensin-converting enzyme,ACE)抑制活性的多肽LEPIGVVGHI,其在调节血压的功能性食品和药物中的应用。
背景技术
高血压作为最常见的心脑血管疾病之一,是脑卒中、冠心病等心血管疾病最重要的危险因素。随着社会经济的高速发展,人们生活饮食结构的改变,高血压的发病率也逐年增高,严重影响为我国居民身体健康。预防、治疗高血压是当今社会面临的主要问题。
ACE作为一种多功能酶,广泛存在于人体中,通过作用于体内的肾素-血管紧张素系统(Renin-Angiotensin System,RAS)和激肽释放酶-激肽系统(Kallikrein-KininSystem,KKS)起到调节血压的功能。因此ACE抑制剂作为一种降血压药物被广泛利用。但由于化学合成西药类降压药物对于人体具有毒副作用,尤其药物吸收对于肝肾组织造成极大负担,引起肝肾损伤,并且停药后症状反复,愈后疗效不十分理想。
生物活性肽是近年来的研究热点。天然蛋白在蛋白酶及微生物作用下会释放出不同结构的肽,研究表明生物活性肽表现出各种生物活性如降血压、降血糖、提高免疫力、抗菌、抗氧化等等。利用天然蛋白质制备ACE抑制剂具有功能明确、安全性高的特点,因此无论作为功能食品还是药品具有广阔的前景。
本发明就是有关于发酵枸杞蛋白制备的一种ACE抑制活性肽。
发明内容
本发明的目的是关于多肽LEPIGVVGHI对于ACE抑制活性;其具有序列表SEQ IDNO:1中氨基酸序列;具有良好的ACE抑制活性,通过抑制ACE活性起到降血压的功能。并且具有开发成为调节血压的功能性食品或者是药物的良好应用前景。
为实现上述目的,本发明在充分利用枸杞蛋白的基础上,以产物多肽LEPIGVVGHI为主要成分,抑制ACE的活性并且调节血压。在此基础上,多肽LEPIGVVGHI可以作为ACE抑制剂、调节血压的药物和相关功能性成品的主要成分。
具有抑制ACE活性和降血压活性的多肽LEPIGVVGHI,氨基酸序列为Leu-Glu-Pro-Ile-Gly-Val-Val-Gly-His-Ile,为单链线性结构,白色粉末状,易溶于水,分子量为1033.24Da;对ACE活性具有较强抑制作用,半抑制浓度(IC50)为674.06μM。
使用生物信息学软件Peptide(http://pepsite2.russelllab.org/)对于多肽LEPIGVVGHI与ACE的结合位点进行预测,蛋白ACE(PDB code:1o8a)的三维结构来自ProteinData Bank(https://www.rcsb.org/)。多肽和蛋白质之间是否存在相互作用通过结果产生的P值和结合位点表示,P值越小则多肽和蛋白质结合可能性越大,若P值>0.5,理论上多肽和蛋白质没有结合可能性。结合位点表示多肽与蛋白质结合位点,若多肽同蛋白质的活性位点相结合,则可以抑制蛋白质同其他蛋白质之间的性互作用,从而抑制该蛋白质活性。
多肽LEPIGVVGHI的p值为0.002975,结合位点为Trp279,Gln281#,His353#,Ala354#,Ser355,Ala356,Trp357,Asp358,Tyr360,His383#,Glu384#,Phe391,Glu411#,Phe457,Phe460,Lys511#,His513#,Tyr520#,Tyr523#,Phe527其中带有#标记的为文献中报道的ACE的重要的活性位点和结合位点,表明多肽LEPIGVVGHI具有良好的ACE抑制活性。
此外生物活性肽的构效关系与ACE的抑制活性相关,多肽LEPIGVVGHI具备ACE抑制剂所要求的特征,多肽中疏水、芳香族氨基酸对ACE的抑制活性有强烈影响。ACE抑制肽更倾向于多肽中含有疏水、芳香族氨基酸。多肽LEPIGVVGHI的中疏水、芳香族氨基酸占总氨基酸数80%,符合要求。
本发明具有如下优点:
本发明从发酵枸杞蛋白中得到了ACE抑制肽,多肽LEPIGVVGHI具有良好的ACE抑制活性,半抑制浓度(IC50)为674.06μM。为ACE抑制肽提供了更加多样的来源,为ACE抑制肽提供了更多的研究参考。
具体实施方式
实施例1多肽LEPIGVVGHI的鉴定
将枸杞蛋白经过枯草芽孢杆菌发酵、酸沉淀和乙醇沉淀以及LC-MS/MS分析,通过生物信息学和构效关系筛选对于ACE有抑制活性的生物活性肽。
其具体方法如下:
(1)枸杞蛋白提取物的制备
以10g干枸杞为原料,称取枸杞并加入100mL去离子水浸泡2小时后粉碎,超声提取60min,于5000rpm离心10min,9.6g沉淀用于提取枸杞蛋白。
沉淀冷冻干燥后再次粉碎得到枸杞粉,枸杞粉中加入192mL的有机溶剂提取液,正己烷:乙醇=2.6:1(v/v),于温度50℃、转速150rpm搅拌提取1小时,过滤,滤饼再用与上述相同的有机溶剂提取液提取一次,提取两次后过滤,滤饼冷冻干燥后为7.5g枸杞蛋白。
(2)枸杞活性肽的制备
将枸杞蛋白加入112mL离子水,用1M的氢氧化钠溶液调节pH至7.5,接种微生物菌种(枯草芽孢杆菌),菌含量为2*108CFU/mL,于37℃发酵48小时;发酵结束后在13000rpm离心15min,取上清液冷冻干燥,得到1g发酵粗提取物。
发酵粗提取物中加入5mL的0.01M的盐酸,在2℃匀浆8min后,于12000rpm离心20min,取上清液加入3倍体积的无水乙醇,4℃静置24小时,于12000rpm离心20min,取上清冷冻干燥,得到0.383g枸杞多肽。
将枸杞多肽用LTQ Orbitrap Velos进行质谱分析:将枸杞多肽除盐,C18SPE预处理柱,经2mL乙腈活化,再用2mL的0.1%(v/v)TFA溶液洗去乙腈。样品用50%(v/v)TFA溶液调节p H值至2后过柱。经2mL的0.1%(v/v)TFA液脱盐后,用1mL 80%(v/v)乙腈/0.1%(v/v)TFA溶液分3次洗脱,洗脱液冷冻干燥后于-20℃保存,用于质谱分析。将冻干样品加适量0.1%(v/v)甲酸复溶,配制成0.5μg/μL的溶液,使用线性离子阱静电场轨道阱组合质谱仪(LTQ Orbitrap Velos)对样品进行质谱分析,设置离子传输毛细管的温度250℃,电喷雾电压2.2kV,归一化碰撞能量35.0%。均使用数据依赖模式(data-dependent mode)对MS和MS/MS进行图谱采集。质谱扫描条件设定为:从每次m/z=400~2000的全扫描中选择10个最高丰度离子峰进行MS/MS扫描。样品平行分析三次,取三次分析鉴定到的共同肽段进行统计。将采集的*.raw文件数据用Thermo Proteome Discoverer Daemon(v1.4)转换成*.mgf格式,再用Mascot(version 2.3.0,Matrix Science,London,UK)谱图在茄科植物蛋白库solanaceae.fasta(https://www.uniprot.org/)中进行检索。三次分析共鉴定到来源于14个不同蛋白的75条共同肽段,41.33%的肽段来自蛋白Fibrillin,肽段中氨基酸数量在8~27之间,计算分子质量在920.4491~2742.4664(Da),表一为部分枸杞多肽的质谱鉴定结果。
表一枸杞多肽的质谱鉴定结果
根据发明内容中关于构效关系的阐述,对鉴定到的共同肽段进行筛选,获得来源于Aldehyde dehydrogenase family 2member C4蛋白的序列为LEPIGVVGHI的多肽。
实施例2多肽LEPIGVVGHI的ACE抑制活性检测
多肽LEPIGVVGHI由南京杰肽生物科技有限公司合成,氨基酸序列为Leu-Glu-Pro-Ile-Gly-Val-Val-Gly-His-Ile,为单链线性结构,白色粉末状,易溶于水,分子量为1033.24Da。
SEQ ID No.1的信息
(a)序列特征
*长度:10氨基酸
*类型:氨基酸
*链型:单链
(b)分子类型:蛋白
序列描述:
SEQ ID No.1
LEPIGVVGHI
N-(3-(2-呋喃酰)丙烯酰-苯氨酰-谷氨酰-谷氨酸(FAPGG,λmax=340nm,ε=2270M-1cm-1,分子量399.40)可以被ACE酶解成N-[3-(呋喃)丙稀醇酰]-2-苯丙氨酸(FAP,λmax=340nm,ε=1512M-1cm-1)和双甘氨肽(GG,在340nm处无吸收),因此可以作为ACE的模拟底物。1mM FAPGG完全转化成FAP和GG的吸光值为0.758,因此可根据340nm吸光度的变化值计算抑制率。
反应体系
(1)缓冲溶液:0.1M PBS缓冲液(pH=8.2,含300mM NaCl)
(2)底物溶液:使用上述缓冲液配制浓度为1.6mM的FAPGG溶液。
(3)酶溶液:使用上述缓冲溶液将ACE配制成0.2U/mL的溶液。
(4)样品溶液:按照实验需要将多肽LEPIGVVGHI用上述缓冲液配制成1.0,0.5,0.1mg/mL浓度的溶液。
实验在96孔板中进行。按照表二依次加入样品溶液、ACE溶液和底物溶液,混合均匀,立即用酶标仪于340nm波长下测定吸光度,记为OD0,37℃温育30min后再次于340nm波长下测定吸光度,记为OD1。测定各个样品的孔的吸光度,令ΔOD=OD0-OD1。每个样品平行测定3次。
表二ACE抑制活性加样方法
“-”代表加入该列等体积的PBS缓冲液
ACE抑制率计算公式:
I=[(ΔODControl-ΔODSample)/(ΔODControl-ΔODblank)]*100%
ΔODcontrol表示反应中样品溶液被等量的缓冲液取代后吸光度的变化;ΔODSample表示反应中样品溶液的吸光度变化;而ΔODblank表示反应中样品溶液、酶溶液被等量的缓冲液取代后吸光度的变化
表三ACE抑制活性实验的吸光度
表四 不同浓度的LEPIGVVGHI对ACE的抑制活性
按上述方法对不同浓度多肽LEPIGVVGHI进行ACE抑制活性检测。表三为不同浓度LEPIGVVGHI对于ACE抑制活性实验的吸光度,由此表四为计算出不同浓度的LEPIGVVGHI对ACE的抑制活性,由此计算多肽LEPIGVVGHI半抑制浓度(IC50)为674.06±146.76μM。
序列表
<110> 中国科学院大连化学物理研究所
<120> 一种枸杞ACE 抑制肽和衍生多肽及应用、混合物
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Leu Glu Pro Ile Gly Val Val Gly His Ile
1 5 10
Claims (6)
1.一种枸杞ACE 抑制肽,其特征在于:所述抑制肽LEPIGVVGHI为序列表SEQ ID NO:1中氨基酸序列;该多肽的氨基酸序列具体为Leu-Glu-Pro-Ile-Gly-Val-Val-Gly-His-Ile。
2.一种权利要求1所述的肽在制备血管紧张素转换酶(angiotensin-convertingenzyme, ACE)抑制剂和/或降血压药物中的应用。
3.按照权利要求2 所述的应用,血管紧张素转换酶(angiotensin-convertingenzyme, ACE)抑制剂和/或降血压药物是以抑制肽LEPIGVVGHI为活性成分 , 其中还添加药物学上可接受的载体或辅料。
4.一种权利要求1所述的抑制肽作为活性成分 在制备体外血管紧张素转换酶(angiotensin-converting enzyme, ACE)抑制剂中的应用,或作为活性成分 在制备具有降血压的功能性食品中的应用。
5.按照权利要求 4 所述的应用,所述抑制剂或功能性食品中还添加药物学上可接受的载体或辅料。
6.一种混合物,为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性食品,其是以抑制肽LEPIGVVGHI为活性成分 , 其中添加药物学上可接受的载体或辅料。
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