CN111499691B - Ace抑制肽p1、其应用及其制备方法 - Google Patents
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Abstract
本发明公开了ACE抑制肽P1其氨基酸序列为:FNLRMQ,本发明还公开了ACE抑制肽P1在辅助降血压药物或辅助降血压功能食品中的应用,以及ACE抑制肽P1的制备方法。本发明得到的ACE抑制肽P1从河鲀鱼皮中提取得到,与ACE酶的结合能力强、活性高,可以有效地用于降血压药物或功能食品中,以辅助降血压,具有特异性高、毒副作用小的优点。
Description
技术领域
本发明涉及生物技术领域,具体地,涉及一种ACE抑制肽P1。本发明还涉及了此ACE抑制肽的应用及其制备方法。
背景技术
人体的血压调节主要是受到血管紧张素***(renin-angiotensin system,RAS)和激肽***(kallikrein kinin system,KKS)控制,而血管紧张素转化酶(Angiotensinconverting enzyme,ACE)在这两个***中起着关键作用。ACE是一种羧基二肽酶,广泛分布于哺乳动物的组织中,主要以膜结合胞外酶的形式存在于血管上皮细胞。在RAS***中,ACE能够使没有生理活性的血管紧张素Ⅰ(AngiotensinⅠ,AngⅠ)转化为具有升压活性的血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)。在KKS***中,ACE可将血管舒缓激肽降解为失活片段,导致血管收缩,引起血压升高。目前高血压的药物治疗主要是对ACE进行抑制,相关研究表明一些从动物、植物以及乳制品中分离得到的天然生物活性肽具有抑制ACE活性、降低血压的作用。相比传统降压药物这些天然活性肽具有特异性高、毒副作用小、疗效好、可用药剂量更大等优点。
对酶解制备ACE抑制肽的研究,有以牛奶、乳酪、大豆、蔬菜、小麦等食物性蛋白为原料,经蛋白酶酶解产生能较好抑制ACE活性的抑制肽制备方法。EIJI etal(Angiotensinconverting enzy me inhibitory activity of the short chain peptides derivedfrom various food pro-teins[J].Nippon Shkuhin Kagaku KogakuKaishi,1996,43(7):
839-840.)水解了12种食物蛋白后发现,从鱼、虾和蟹等水产动物蛋白中酶解制得的ACE抑制肽,其降压活性优于其他食物性蛋白。
ACE抑制肽在母体蛋白中没有活性,但能通过酶水解方法从母体蛋白中释放出。由于ACE抑制肽氨基酸组成和结构差别很大,没有固定或统一氨基酸组成,且食物中蛋白酶解产物成分相当复杂,这样就大大增加对其分离提取难度。
ACE抑制肽活性检测方法主要有体内检测和体外实验二种。体外检测一般使用IC50表示ACE抑制剂抑制活性,抑制物IC50越小,其活性越高。
孙美玲等在文章《海参水煮液中ACE抑制肽的分离纯化》(《大连工业大学学报》2019年1月)中采用大孔树吸附的方法对海参水煮液酶解物多肽进行分离,并应用p18反相硅胶柱和LH-20葡聚糖凝胶柱对活性最高的组分进行ACE抑制肽分离纯化,得到2种多肽单体,ACE抑制活性的IC50分别为0.74和1.77mg/mL。
公开号为CN108129561A的专利中公开了一种ACE抑制肽,将大黄鱼肌动蛋白进行虚拟酶切,得到一定量的肽序列,筛选其中未被报道的三肽序列进行毒性、水溶性和ADMET性质的预测,并利用Discoverystudio软件进行分子对接,最终筛选得到三肽His-Glu-Arg(HER)。利用RP-HPLC法进行三肽HER体外ACE抑制活性的鉴定。结果显示HER具有良好ACE抑制活性,IC50值为1.82±0.06mM。
河鲀为硬骨鱼纲鲀科鱼类的统称,随着河鲀鱼加工利用市场的开放及发展,大量河鲀鱼加工利用后的副产物(如鱼皮、鱼头、鱼内脏等)则作为废弃物丢弃或者加工成动物饲料,不仅将会给将会给环境带来巨大的压力,给社会带来负面效应也是对鱼皮资源极大的浪费。因此,充分利用河鲀鱼其中的营养成分,开发高附加值的各类产品,将会对整个河鲀鱼市场带来新的良性发展。有研究表明鱼皮的蛋白质较于肌肉高,鱼皮中的胶原含量最高可占其蛋白质总量的80%多,且鱼皮的蛋白质除了主要为纤维状的胶原外,还含有少量白蛋白、粘性蛋白、球蛋白、粘蛋白。目前尚未见从河鲀中提取ACE抑制肽的相关报道。
发明内容
本发明的目的在于提供一种从河鲀鱼皮中提取得到的ACE抑制肽P1,与ACE酶的结合能力强、活性高,可以应用于辅助降血压药物或功能食品中。
为实现上述目的,本发明采用以下技术方案:
本发明公开了ACE抑制肽P1,其氨基酸序列为:FNLRMQ。
进一步地,其分子量为807.97Da,IC50为0.47μM。
本发明公开了上述ACE抑制肽P1在辅助降血压药物或辅助降血压功能食品中的应用。
本发明还公开了ACE抑制肽P1的制备方法,包括如下步骤:
S1.取河鲀鱼皮,加入蛋白酶进行酶解,酶解温度控制为50℃~60℃,酶底比为2%~4%,pH为11~12,酶解时间为5h~7h。
S2.对酶解产物进行初步过滤,去除残渣得到澄清的多肽酶解液。
S3.选择聚醚砜超滤膜对多肽酶解液进行超滤分离,将胶原酶解多肽分为若干超滤组分,冻干不同分子量分布的多肽液,得到多肽冻干粉。
S4.称取适量的不同分子量的多肽冻干粉,分别配制成多肽溶液,测定其对ACE抑制率的大小,筛选得到对ACE抑制活性最强的多肽冻干粉。
S5.将筛选得到的多肽冻干粉采用LC-MS/MS进行质谱测定,质谱测定的结果采用质谱分析软件分析,获得若干多肽序列。
S6.通过软件将多肽序列与ACE蛋白进行分子对接,在对接之前通过能量最小化将多肽的2D结构转换为3D结构,筛选得到与ACE蛋白的结合能力强的多肽序列。
S7.将筛选得到的多肽序列进行固相合成,筛选活性高的多肽序列,即得到ACE抑制肽P1。
其中,步骤S1中酶解温度为55℃,酶底比为2%,pH为12,酶解时间为6h。
优选地,步骤S3中选择截留量为1kDa和5kDa的聚醚砜超滤膜对多肽酶解液进行超滤分离,将胶原酶解多肽分为Mw<1kDa,1kDa<Mw<5kDa和Mw>5kDa的三个超滤组分,冻干以上三个不同分子量分布的多肽液,得到多肽冻干粉。
优选地,步骤S5中质谱测定条件为:色谱柱为PepMap RPLC C18,阳离子模式,扫描范围:m/z 300–1500Da,发射极喷雾电压2kV。
本发明具有如下有益效果:本发明方法制备得到的ACE抑制肽P1与ACE酶的亲和力好,活性较高,IC50值为0.47μM,可应用于开发辅助降血压的治疗药物或功能食品中。且由于本发明ACE抑制肽P1是从河鲀鱼皮中提取得到,相比传统降压药物具有特异性高、毒副作用小的优点。
附图说明
图1为不同酶解温度对ACE抑制率的影响。
图2为不同酶解时间对ACE抑制率的影响。
图3为不同酶底比对ACE抑制率的影响。
图4为不同pH对ACE抑制率的影响。
图5为不同分子量大小的多肽组分对ACE抑制率的影响。
图6为不同分子量大小的多肽组分的IC50值。
图7为本发明多肽FNLRMQ的液相色谱图。
图8为本发明多肽FNLRMQ的质谱图。
图9为FNLRMQ与ACE的结合模式示意图。
图10为图9的局部放大示意图。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案,下面结合附图及具体实施例对本发明作进一步详细的描述。
本发明公开了一种ACE抑制肽P1,其氨基酸序列为:FNLRMQ。
ACE抑制肽P1的制备方法,包括如下步骤:
S1.取河鲀鱼皮,加入蛋白酶进行酶解,酶解温度控制为50℃~60℃,酶底比为2%~4%,pH为11~12,酶解时间为5h~7h。优选为:酶解温度为55℃,酶底比为2%,pH为12,酶解时间为6h。
本发明以ACE抑制率为考核指标,根据选用的蛋白酶在不同的酶解时间、酶解温度、pH和酶添加量的条件下进行试验,考察各因素对ACE抑制率的影响。具体实验结果如下详述。
(1)不同酶解温度对ACE抑制率活性的影响
如图1所示,随着酶解温度的提高,胶原酶解产物对ACE抑制活性呈现先增加后下降的趋势,当温度达到65℃时,胶原酶解产物对ACE抑制活性急剧下降,可能是由于温度过高使得蛋白酶失活,造成酶活力的下降,导致水解效果下降,从而降低了其对DPPH的清除率。因此,确定55℃为酶解反应最佳温度。
(2)不同酶解时间对ACE抑制率活性的影响
如图2所示,随着酶解时间的增加,胶原酶解产物对ACE抑制活性的影响也是呈现先增加后降低的趋势,酶解时间过长有活性的肽段可能会被持续酶解,从而使得抑制活性下降。因此,最适宜的酶解时间为6h。
(3)不同酶底比对ACE抑制率活性的影响
如图3可知,随着酶浓度的增加,胶原酶解产物对ACE抑制率呈现为先快速增加而后缓慢增加,在酶底比为4%时达到最高,但其抑制率比2%和3%增加不明显。随着进一步增加酶量,ACE抑制率反而下降,当酶被底物饱和的时候,反应达到平衡。综合考虑经济成本及酶解效果,酶底比为2%较佳。
(4)不同pH对ACE抑制率活性的影响
如图4所示,随着pH的升高,胶原酶解产物对ACE抑制活性的影响也是呈现先增加后降低的趋势,这表明pH过高或过低均会抑制蛋白酶的活性,从而使得酶解效率下降,表现为ACE抑制率下降。因此,选择pH=12为适宜的pH。
S2.对酶解产物进行初步过滤,去除残渣得到澄清的多肽酶解液。初步过滤可以采用八层纱布进行过滤。
S3.选择截留量为1kDa和5kDa的聚醚砜超滤膜对多肽酶解液进行超滤分离,将胶原酶解多肽分为Mw<1kDa,1kDa<Mw<5kDa和Mw>5kDa的三个超滤组分,冻干以上三个不同分子量分布的多肽液,得到多肽冻干粉。
S4.称取适量的不同分子量的多肽冻干粉,分别配制成1mg/mL的多肽溶液,测定其对ACE抑制率的大小,筛选得到对ACE抑制活性最强的多肽冻干粉。
ACE抑制率的测试方法为:将100μL样品多肽溶液与50μL的50mU/mL的ACE(ACE溶于5000μL含有0.3mol/L氯化钠的0.1mol/L硼酸盐缓冲液,pH 8.3),37℃水浴10min。然后加入150μL5mM HHL(用含0.3mol/L氯化钠,pH值8.3的0.1mol/L硼酸盐缓冲液配制),37℃水浴30min,结束加入200μL 1.0mol/L HCl终止反应,蒸馏水作为空白样品。在此过程中,ACE酶解HHL释放出马尿酸,马尿酸的浓度可通过高效液相色谱UV检测器228nm处进行检测。其中ACE抑制率公式为:
其中,ACK为空白样品马尿酸的峰面积,AS为酶解产物的马尿酸的峰面积。
由图5、图6的结果可知,Mw<1kDa的抑制率最佳,1kDa<Mw<5kDa的次之,Mw>5kDa对ACE的抑制率最弱。
S5.将筛选得到的Mw<1kDa的组分多肽冻干粉采用LC-MS/MS进行质谱测定,质谱测定的结果采用质谱分析软件分析,获得若干多肽序列。
质谱测定条件为:色谱柱为PepMap RPLC C18,阳离子模式,扫描范围:m/z 300–1500Da,发射极喷雾电压2kV。质谱检测的结果采用PEAKS STUDIO软件分析,获得多肽序列82条。
S6.通过MOE软件将多肽序列与ACE蛋白进行分子对接,在对接之前通过能量最小化将多肽的2D结构转换为3D结构,筛选得到与ACE蛋白的结合能力强的多肽序列。
蛋白质ACE的3D结构可从RCSB蛋白质数据库(PDB ID:1O8A)下载。分子对接的结果如下表1所示,对接分值的数值越小(绝对值越大)表明多肽与ACE酶的结合能力越强,也最越有可能抑制ACE酶的活性。
表1不同多肽序列与ACE酶的分子亲和力大小
S7.将筛选得到的多肽序列进行固相合成,筛选活性高的多肽序列,即得到ACE抑制肽P1。
将表1的多肽序列固相合成,筛选得到活性较高的多肽序列,即上表中的14,氨基酸序列号为FNLRMQ,即得到本发明ACE抑制肽P1。对ACE抑制肽P1进行液相和质谱的测定,如图7、图8所示,分子量为807.97Da,IC50值为0.47μM。ACE抑制肽P1可溶解于超纯水、PBS和DMSO,如表2所示。
表2.ACE抑制肽P1的溶解性测试
溶剂 | 溶解性 | 多肽浓度 |
超纯水 | 可溶 | ≦10mg/ml |
1X DPBS*(pH 7.1±0.1) | 可溶 | ≦10mg/ml |
DMSO | 可溶 | ≦10mg/ml |
将本发明的多肽通过MOE软件与ACE蛋白进行分子对接模拟实验,可得本发明ACE抑制肽P1与ACE的结合方式如图9、图10所示。ACE中H353的咪唑基的氮原子与P1中的M5的硫醚基的硫原子形成一个氢键。ACE中D453的羧基氧原子与P1中F1骨架的氮原子形成一个氢键。ACE中K454氨基的氮原子与P1中N2酰胺基的氧原子形成一个氢键。ACE中Y523的酚羟基的氧原子与P1中Q6的酰胺基的氧原子形成一个氢键。ACE中E162的羧基氧原子与P1中R4的胍基氮原子形成盐桥。ACE中D377的羧基氧原子与P1中R4的胍基氮原子形成盐桥。ACE中E376的羧基氧原子与P1中R4的胍基氮原子形成盐桥。ACE中的Zn2+与P1中Q6的酰胺基的氧原子形成离子接触。
对接模拟研究表明ACE中的Zn2+,E162,H353,E376,D377,D453,K454和Y523参与与F1,N2,R4,M5和Q6的结合,P1的残余物通过离子接触,盐桥结合和氢键相互作用。如下表3所示。
表3.ACE抑制肽P1与ACE活性接触列表
链A | 残基 | 链B | 残基 | 相互作用类型 |
ACE | His353 | P1 | M5 | 氢键相互作用 |
ACE | Asp453 | P1 | F1 | 氢键相互作用 |
ACE | Lys454 | P1 | N2 | 氢键相互作用 |
ACE | Tyr523 | P1 | Q6 | 氢键相互作用 |
ACE | Zn2+ | P1 | Q6 | 离子键 |
ACE | Glu162 | P1 | R4 | 盐桥 |
ACE | Glu376 | P1 | R4 | 盐桥 |
ACE | Asp377 | P1 | R4 | 盐桥 |
综上,通过本发明方法制备得到的ACE抑制肽P1与ACE酶的相互作用强,活性高,可以应用于辅助降血压药物或辅助降血压功能食品中。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
序列表
<110> 福建省水产研究所(福建水产病害防治中心)
<120> ACE抑制肽P1、其应用及其制备方法
<160> 1
<170> PatentIn version 3.5
<210> P1
<211> 6
<212> PRT
<213> 河鲀
<400> P1
Phe Asn Leu Arg Met Gln
1 5
Claims (3)
1.ACE抑制肽P1,其特征在于,其氨基酸序列为:FNLRMQ。
2.如权利要求1所述的ACE抑制肽P1,其特征在于,其分子量为807.97Da,IC50为0.47μM。
3.权利要求1所述的ACE抑制肽P1在制备辅助降血压药物中的应用。
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