CN107163130B - 一种血管紧张素转化酶抑制肽及其制备提取方法 - Google Patents
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Abstract
本发明公开一种血管紧张素转化酶抑制肽及其制备提取方法,该血管紧张素转化酶抑制肽氨基酸序列为:Leu‑Leu‑Tyr‑Gln‑Glu‑Pro‑Val‑Leu‑Gly‑Pro‑Val‑Pro‑Arg;本发明制备出一种限进亲和介质,利用该限进亲和介质从酪蛋白的酶解液中分离提取目标小分子肽,然后对该限进亲和介质进行洗脱,再用HPLC从洗脱液中提取出血管紧张素转化酶抑制肽。本发明适用于从复杂的生物基质样品中快速、有效的分离出小分子活性物,提高分离效果。
Description
技术领域
本发明涉及一种血管紧张素转化酶抑制肽及其制备提取方法。
背景技术
高血压是一种世界性的疾病,影响到大多数国家30%的成年人口。它是最常见的严重慢性疾病,是引发动脉硬化,中风,心肌梗死和终末期肾脏疾病等的重要因素。血管紧张素转化酶(angiotensin converting enzyme,ACE)是调节外周血压和电解质平衡的关键酶,它能促进血管紧张素I高度有效的转化为血管紧张素II,导致血压升高。ACE抑制剂(angiotensin converting enzyme inhibitor,ACEI)可以抑制ACE的活性,通过动物模型和临床实验已证明其可有效的降低血压。从天然产物中提取的ACE抑制肽以其稳定、安全、无副作用的优势已经引起了广泛的兴趣,快速、高效的从天然产物中纯化ACE抑制肽将对治疗高血压有着非常重要的作用。但小分子肽所处环境复杂且浓度很低,特别是杂质特性与目标小分子肽性质相似,使得小分子肽的分离纯化非常困难。
传统的分离方法如超滤、微滤、盐析、透析、离子交换、电泳等操作过程复杂、分离速度慢、样品处理量小、回收率低以及成本昂贵。另一方面,由于生物样品中目标小分子含量低,大量存在的生物大分子会影响小分子扩散和竞争吸附位点,会降低目标小分子的纯度及吸附量。因此高选择性并高效的分离纯化方法是小分子肽的重要研究方向。
发明内容
本发明的目的是提供一种血管紧张素转化酶抑制肽及其制备提取方法,本发明先利用聚乙二醇单甲醚5000将固定化金属亲和介质(IMAC)进行修饰制备限进亲和介质这种分离介质,限进亲和介质具有的特异性吸附和大分子阻拒的特点,再利用限进亲和介质高效快速地提取分离血管紧张素转化酶抑制肽,提高了IMAC分离小分子肽的效率。
为解决上述技术问题,本发明采用如下技术方案:
一种血管紧张素转化酶抑制肽,其氨基酸序列为:Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg。
一种血管紧张素转化酶抑制肽的制备提取方法,其特征在于,包括以下步骤:
S1.磁性二氧化硅微球的制备:取0.03~0.05g Fe3O4、0.95~1.05mL正硅酸乙酯(TEOS)和0.08~0.12mL聚乙二醇单辛基苯基醚(Triton X-100)加入100mL乙醇/水混合液中并以50~60KHz功率超声分散8~15min,然后加入9mL浓度为25%的氨水,反应2~3h后利用外加磁场分离出固体产品得到磁性二氧化硅微球,所述Fe3O4的粒径为10~30nm,所述乙醇/水混合液中乙醇和水的体积比为1:1;Fe3O4为磁性粒子,Triton X-100为表面活性剂,使得Fe3O4粒子分布均匀,氨水给反应提供碱性环境,正硅酸乙酯在碱性条件下能够水解生成二氧化硅,反应结束后得到磁性二氧化硅微球;
S2.限进亲和介质的制备:利用3-氨丙基三乙氧基硅烷(APTES)将S1中的磁性二氧化硅微球进行氨基化改性得到氨基化微球,再用环氧氯丙烷对氨基化微球进行活化,然后用活化后的氨基化微球螯合铜离子得到螯合微球;将1~2g聚乙二醇单甲醚5000(mPEG-5000)加入装有4~8mL二甲基亚砜(DMSO)的三口瓶中,再向三口瓶中加入1.5~3mL氯仿及0.6~0.8mL乙酸酐并在35~40℃反应12h,得到mPEG-CHO;将0.2g的螯合微球溶于50~100mL乙醇和水混合溶液中,乙醇和水的体积比为1:1,加入1g的mPEG-CHO,在室温下搅拌反应24h后再加入0.05~0.08g还原剂NaBH3,继续反应48h,将反应后产物置于透析袋中,在蒸馏水中透析48h,最后磁性分离得到限进亲和介质;磁性二氧化硅微球氨基化后能接枝mPEG-CHO,氨基化微球与铜离子螯合使得微球获得特异性吸附功能;
S3.将酪蛋白用胰蛋白酶和胃蛋白酶在55℃下酶解2h得到酶解液,所述胰蛋白酶和胃蛋白酶的质量比为1:1,所述胰蛋白酶和胃蛋白酶组成的复合蛋白酶与酪蛋白的质量比为1:60~100,然后采用超滤离心管(10KDa)对酶解液进行超滤得到滤液,将10mg S2中的限进亲和介质加入2mL滤液中进行吸附1~2h,然后利用外加磁场分离出限进亲和介质,再用0.5~1mL浓度为0.5~1mol/L的氯化铵和0.5~1mL浓度为0.5~1mol/L的氯化钠对其洗脱0.5~1h得到洗脱液,旋转蒸发洗脱液得到浓缩液;酶解液超滤是为了将一部分杂质和没有酶解的大分子除去,使得酶解液得到纯化,有利于限进亲和介质吸附目标小分子多肽;
S4.采用HPLC对S3中所述浓缩液进行分离,流动相为:A相为水(1%TF A),B相为乙腈(1%TFA);分离程序为:0~40min,B相乙腈体积由5%到30%,流速为0.5mL/min,检测波长为280nm,分离收集到氨基酸序列为Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg的血管紧张素转化酶抑制肽。
作为优选,S2中所述氨基化微球的制备方法为:取0.2g S1中的磁性二氧化硅微球置于三口瓶中,加入200mL乙醇,2mL去离子水,然后超声分散30min,再以400~600r/min的转速搅拌10min,向三口瓶中逐滴加入1.8~2.2mLAPTES,在72~78℃下回流8~10h,反应结束后用磁铁在瓶底吸附,倾去上清液,再加入无水乙醇,经超声、洗涤,最后磁性分离得到氨基化微球。
作为优选,S2所述螯合微球的制备方法为:称取0.15g S2中的氨基化微球,加入2.5mL浓度为0.4mol/L的NaOH和2.5mL浓度为2.5mol/L环氧氯丙烷,接着加入0.9~1.1mL的DMSO,在30~40℃下摇床振荡4h,然后去离子水洗涤去除未反应的环氧氯丙烷得到环氧化微球,将制备出的环氧化微球加入20mL浓度为0.5mol/L的Na2CO3溶液中,再加入0.05~0.1g亚氨基二乙酸(IDA),在40~50℃下反应8h,Cu2+与IDA发生配位反应,反应结束后加入20mL浓度为0.05mol/L的CuSO4溶液,摇床震荡2h得到螯合微球。
,作为优选,所述限进亲和介质的Cu2+螯合密度不低于50μmol/g,保障限进亲和介质对小分子肽特异性吸附性能;mPEG-5000的接枝率为10.3%~14.2%,接枝率太低无法形成体积排阻效应,接枝率过高则会不利于小分子肽的吸附。
本发明提供所述血管紧张素转化酶抑制肽在制备减缓高血压症状的功能食品中的应用。
本发明的有益效果:
本发明制备的限进亲和介质能特异性吸附小分子多肽的同时阻拒大分子杂蛋白的吸附,相对于IMAC其活性组分的含量提高20%~30%。利用制备的限进亲和介质快速的从酪蛋白酶解液中分离纯化的ACE抑制肽具有良好的ACE抑制效果。本发明制备的限进亲和介质非常适用于从复杂的生物基质样品中快速、有效的分离出小分子活性物,提高分离效果。
附图说明
图1为实施例1中酪蛋白酶解液(a)、亲和介质洗脱液(b)和限进亲和介质洗脱液(c)的凝胶色谱图。测试条件:色谱柱:Shodex PTOTENIN KW-802.5,检测器:DAD二极管阵列检测器,流动相:0.01M PBS缓冲液(pH=7),检测波长:280nm,流速:0.5mL/min;
图2为实施例1中酪蛋白酶解液(a)、亲和介质洗脱液(b)和限进亲和介质洗脱液(c)的高效液相色谱图。测试条件:色谱柱:Agilent SB-C18,检测器:DAD二极管阵列检测器,流动相:A相:水(0.1%TFA),B相:乙腈(0.1%TFA),流动相:5%~30%B相(0~40min),流速:0.5mL/min,检测波长:280nm;
图3为实施例1中限进亲和介质从酪蛋白酶解液中纯化的多肽Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg的质谱图。
具体实施方式
参照附图对本发明具体实施例做进一步说明。
本发明的血管紧张素转化酶抑制肽的氨基酸序列为:Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg。
实施例1
一种血管紧张素转化酶抑制肽的制备提取方法,包括以下步骤:
(1)磁性二氧化硅微球的制备:取0.03g平均粒径为10nm的Fe3O4、1mL正硅酸乙酯(TEOS)和0.1mL聚乙二醇单辛基苯基醚(Triton X-100)加入100mL乙醇/水混合液中,乙醇和水的体积比为1:1,并以50KHz功率超声分散10min,然后加入9mL浓度为25%的氨水,反应2h后利用外加磁场分离出固体产品得到磁性二氧化硅微球;
(2)氨基化微球的制备:取0.2g(1)中制备的磁性二氧化硅微球置于三口瓶中,加入200mL乙醇,2mL去离子水,然后超声分散30min,再以500r/min的转速搅拌10min,向三口瓶中逐滴加入2mLAPTES,在75℃下回流8h,反应结束后用磁铁在瓶底吸附,倾去上清液,再加入无水乙醇,经超声、洗涤,磁性分离得到氨基化微球;
(3)螯合微球的制备:称取0.15g(2)中制备的氨基化微球,加入2.5mL浓度为0.4mol/L的NaOH和2.5mL浓度为2.5mol/L环氧氯丙烷,接着加入1mL的DMSO,在30℃下摇床振荡4h,然后去离子水洗涤得到环氧化微球,将制备出的环氧化微球加入20mL浓度为0.5mol/L的Na2CO3溶液中,再加入0.05g亚氨基二乙酸(IDA),在50℃下反应8h,反应结束后加入20mL浓度为0.05mol/L的CuSO4溶液,摇床震荡2h得到螯合微球;
(4)限进亲和介质的制备:将1g聚乙二醇单甲醚5000(mPEG-5000)加入装有4mL二甲基亚砜(DMSO)的三口瓶中,再向三口瓶中加入1.5mL氯仿及0.6mL乙酸酐并在40℃反应12h,得到mPEG-CHO;将0.2g(3)中制备的的螯合微球溶于50mL乙醇和水(乙醇和水的体积比为1:1)混合溶液中,加入1g的mPEG-CHO,在室温下搅拌反应24h后再加入0.05g还原剂NaBH3,继续反应48h,将反应后产物置于透析袋中,在蒸馏水中透析48h,最后磁性分离得到限进亲和介质。
(5)将60g酪蛋白用1g胰蛋白酶和1g胃蛋白酶在55℃下酶解2h得到酶解液,然后采用超滤离心管(10KDa)对酶解液进行超滤得到滤液,将10mg(4)中制备的限进亲和介质加入2mL滤液中进行吸附2h,然后利用外加磁场分离出限进亲和介质,再用0.5mL浓度为0.5mol/L的氯化铵和0.5mL浓度为0.5mol/L的氯化钠对其洗脱1h得到洗脱液,旋转蒸发洗脱液得到浓缩液;
(6)采用HPLC对(5)中浓缩液进行分离,流动相为:A相为水(1%TFA),B相为乙腈(1%TFA);分离程序为:0~40min,B相乙腈体积由5%到30%,流速为0.5mL/min,检测波长为280nm,分离收集到氨基酸序列为Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg的血管紧张素转化酶抑制肽。
所述限进亲和介质的Cu2+螯合密度为52μmol/g,mPEG-5000的接枝率为10.3%。
实施例2
一种血管紧张素转化酶抑制肽的制备提取方法,包括以下步骤:
(1)磁性二氧化硅微球的制备:取0.04g平均粒径为15nm的Fe3O4、1.05mL正硅酸乙酯(TEOS)和0.12mL聚乙二醇单辛基苯基醚(Triton X-100)加入100mL乙醇/水混合液中,乙醇和水的体积比为1:1,并以60KHz功率超声分散15min,然后加入9mL浓度为25%的氨水,反应3h后利用外加磁场分离出固体产品得到磁性二氧化硅微球;
(2)氨基化微球的制备:取0.2g(1)中制备的磁性二氧化硅微球置于三口瓶中,加入200mL乙醇,2mL去离子水,然后超声分散30min,再以400r/min的转速搅拌10min,向三口瓶中逐滴加入1.8mLAPTES,在72℃下回流10h,反应结束后用磁铁在瓶底吸附,倾去上清液,再加入无水乙醇,经超声、洗涤,磁性分离得到氨基化微球;
(3)螯合微球的制备:称取0.15g(2)中制备的氨基化微球,加入2.5mL浓度为0.4mol/L的NaOH和2.5mL浓度为2.5mol/L环氧氯丙烷,接着加入0.9mL的DMSO,在40℃下摇床振荡4h,然后去离子水洗涤得到环氧化微球,将制备出的环氧化微球加入20mL浓度为0.5mol/L的Na2CO3溶液中,再加入0.08g亚氨基二乙酸(IDA),在40℃下反应8h,反应结束后加入20mL浓度为0.05mol/L的CuSO4溶液,摇床震荡2h得到螯合微球;
(4)限进亲和介质的制备:将2g聚乙二醇单甲醚5000(mPEG-5000)加入装有8mL二甲基亚砜(DMSO)的三口瓶中,再向三口瓶中加入2.8mL氯仿及0.8mL乙酸酐并在38℃反应12h,得到mPEG-CHO;将0.2g(3)中制备的的螯合微球溶于100mL乙醇和水(乙醇和水的体积比为1:1)混合溶液中,加入1g的mPEG-CHO,在室温下搅拌反应24h后再加入0.08g还原剂NaBH3,继续反应48h,将反应后产物置于透析袋中,在蒸馏水中透析48h,最后磁性分离得到限进亲和介质。
(5)将80g酪蛋白用1g胰蛋白酶和1g胃蛋白酶在55℃下酶解2h得到酶解液,然后采用超滤离心管(10KDa)对酶解液进行超滤得到滤液,将10mg(4)中制备的限进亲和介质加入2mL滤液中进行吸附1.5h,然后利用外加磁场分离出限进亲和介质,再用1mL浓度为1mol/L的氯化铵和1mL浓度为1mol/L的氯化钠对其洗脱0.5h得到洗脱液,旋转蒸发洗脱液得到浓缩液;
(6)采用HPLC对(5)中浓缩液进行分离,流动相为:A相为水(1%TFA),B相为乙腈(1%TFA);分离程序为:0~40min,B相乙腈体积由5%到30%,流速为0.5mL/min,检测波长为280nm,分离收集到氨基酸序列为Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg的血管紧张素转化酶抑制肽。
所述限进亲和介质的Cu2+螯合密度为61μmol/g,mPEG-5000的接枝率为12.4%。
实施例3
一种血管紧张素转化酶抑制肽的制备提取方法,包括以下步骤:
(1)磁性二氧化硅微球的制备:取0.05g平均粒径为30nm的Fe3O4、0.95mL正硅酸乙酯(TEOS)和0.08mL聚乙二醇单辛基苯基醚(Triton X-100)加入100mL乙醇/水混合液中,乙醇和水的体积比为1:1,并以55KHz功率超声分散8min,然后加入9mL浓度为25%的氨水,反应2.5h后利用外加磁场分离出固体产品得到磁性二氧化硅微球;
(2)氨基化微球的制备:取0.2g(1)中制备的磁性二氧化硅微球置于三口瓶中,加入200mL乙醇,2mL去离子水,然后超声分散30min,再以600r/min的转速搅拌10min,向三口瓶中逐滴加入2.2mLAPTES,在78℃下回流9h,反应结束后用磁铁在瓶底吸附,倾去上清液,再加入无水乙醇,经超声、洗涤,磁性分离得到氨基化微球;
(3)螯合微球的制备:称取0.15g(2)中制备的氨基化微球,加入2.5mL浓度为0.4mol/L的NaOH和2.5mL浓度为2.5mol/L环氧氯丙烷,接着加入1.1mL的DMSO,在35℃下摇床振荡4h,然后去离子水洗涤得到环氧化微球,将制备出的环氧化微球加入20mL浓度为0.5mol/L的Na2CO3溶液中,再加入0.1g亚氨基二乙酸(IDA),在45℃下反应8h,反应结束后加入20mL浓度为0.05mol/L的CuSO4溶液,摇床震荡2h得到螯合微球;
(4)限进亲和介质的制备:将1.6g聚乙二醇单甲醚5000(mPEG-5000)加入装有6mL二甲基亚砜(DMSO)的三口瓶中,再向三口瓶中加入3mL氯仿及0.7mL乙酸酐并在35℃反应12h,得到mPEG-CHO;将0.2g(3)中制备的的螯合微球溶于80mL乙醇和水(乙醇和水的体积比为1:1)混合溶液中,加入1g的mPEG-CHO,在室温下搅拌反应24h后再加入0.07g还原剂NaBH3,继续反应48h,将反应后产物置于透析袋中,在蒸馏水中透析48h,最后磁性分离得到限进亲和介质。
(5)将100g酪蛋白用1g胰蛋白酶和1g胃蛋白酶在55℃下酶解2h得到酶解液,然后采用超滤离心管(10KDa)对酶解液进行超滤得到滤液,将10mg(4)中制备的限进亲和介质加入2mL滤液中进行吸附1h,然后利用外加磁场分离出限进亲和介质,再用0.7mL浓度为0.8mol/L的氯化铵和0.8mL浓度为0.7mol/L的氯化钠对其洗脱0.6h得到洗脱液,旋转蒸发洗脱液得到浓缩液;
(6)采用HPLC对(5)中浓缩液进行分离,流动相为:A相为水(1%TFA),B相为乙腈(1%TFA);分离程序为:0~40min,B相乙腈体积由5%到30%,流速为0.5mL/min,检测波长为280nm,分离收集到氨基酸序列为Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg的血管紧张素转化酶抑制肽。
所述限进亲和介质的Cu2+螯合密度为57μmol/g,mPEG-5000的接枝率为14.2%。
本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种血管紧张素转化酶抑制肽的制备提取方法,其特征在于,所述血管紧张素转化酶抑制肽的氨基酸序列为:Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-V al-Pro-Arg;
所述的血管紧张素转化酶抑制肽的制备提取方法,包括以下步骤:
S1.磁性二氧化硅微球的制备:取0.03~0.05g Fe3O4、0.95~1.05mL正硅酸乙酯(TEOS)和0.08~0.12mL聚乙二醇单辛基苯基醚(Triton X-100)加入100mL乙醇/水混合液中并以50~60KHz功率超声分散8~15min,然后加入9mL浓度为25%的氨水,反应2~3h后利用外加磁场分离出固体产品得到磁性二氧化硅微球,所述Fe3O4的粒径为10~30nm,所述乙醇/水混合液中乙醇和水的体积比为1:1;
S2.限进亲和介质的制备:利用3-氨丙基三乙氧基硅烷(APTES)将S1中的磁性二氧化硅微球进行氨基化改性得到氨基化微球,再用环氧氯丙烷对氨基化微球进行活化,然后用活化后的氨基化微球螯合铜离子得到螯合微球;将1~2g聚乙二醇单甲醚5000(mPEG-5000)加入装有4~8mL二甲基亚砜(DMSO)的三口瓶中,再向三口瓶中加入1.5~3mL氯仿及0.6~0.8mL乙酸酐并在35~40℃反应12h,得到mPEG-CHO;将0.2g的螯合微球溶于50~100mL乙醇和水混合溶液中,乙醇和水的体积比为1:1,加入1g的mPEG-CHO,在室温下搅拌反应24h后再加入0.05~0.08g还原剂NaBH3,继续反应48h,将反应后产物置于透析袋中,在蒸馏水中透析48h,最后磁性分离得到限进亲和介质;
S3.将酪蛋白用胰蛋白酶和胃蛋白酶在55℃下酶解2h得到酶解液,所述胰蛋白酶和胃蛋白酶的质量比为1:1,所述胰蛋白酶和胃蛋白酶组成的复合蛋白酶与酪蛋白的质量比为1:60~100,然后采用10KDa超滤离心管对酶解液进行超滤得到滤液,将10mg S2中的限进亲和介质加入2mL滤液中进行吸附1~2h,然后利用外加磁场分离出限进亲和介质,再用0.5~1mL浓度为0.5~1mol/L的氯化铵和0.5~1mL浓度为0.5~1mol/L的氯化钠对其洗脱0.5~1h得到洗脱液,旋转蒸发洗脱液得到浓缩液;
S4.采用HPLC对S3中所述浓缩液进行分离,流动相为:A相为水,内含体积分数为1%的TFA;B相为乙腈,内含体积分数为1%的TFA;分离程序为:0~40min,B相乙腈体积由5%到30%,流速为0.5mL/min,检测波长为280nm,分离收集到氨基酸序列为Leu-Leu-Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Pro-Arg的血管紧张素转化酶抑制肽;
其中,S2所述螯合微球的制备方法为:称取0.15g S2中的氨基化微球,加入2.5mL浓度为0.4mol/L的NaOH和2.5mL浓度为2.5mol/L环氧氯丙烷,接着加入0.9~1.1mL的DMSO,在30~40℃下摇床振荡4h,然后去离子水洗涤得到环氧化微球,将制备出的环氧化微球加入20mL浓度为0.5mol/L的Na2CO3溶液中,再加入0.05~0.1g亚氨基二乙酸(IDA),在40~50℃下反应8h,反应结束后加入20mL浓度为0.05mol/L的CuSO4溶液,摇床震荡2h得到螯合微球。
2.根据权利要求1所述的血管紧张素转化酶抑制肽的制备提取方法,其特征在于,S2中所述氨基化微球的制备方法为:取0.2g S1中的磁性二氧化硅微球置于三口瓶中,加入200mL乙醇,2mL去离子水,然后超声分散30min,再以400~600r/min的转速搅拌10min,向三口瓶中逐滴加入1.8~2.2mLAPTES,在72~78℃下回流8~10h,反应结束后用磁铁在瓶底吸附,倾去上清液,再加入无水乙醇,经超声、洗涤,最后磁性分离得到氨基化微球。
3.根据权利要求1所述的血管紧张素转化酶抑制肽的制备提取方法,其特征在于,所述限进亲和介质的Cu2+螯合密度不低于50μmol/g,mPEG-5000的接枝率为10.3%~14.2%。
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