CN108101964B - 酸乳中新型ace抑制肽及其基因工程生产方法 - Google Patents
酸乳中新型ace抑制肽及其基因工程生产方法 Download PDFInfo
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Abstract
本发明涉及一种酸乳中新型ACE抑制肽及其基因工程生产方法，从干酪乳杆菌(Lactobacillus Casei)HZ1发酵乳中鉴定获得了2个分子量分别为18.3kD、12.2kD的新型ACE抑制肽ACEI1和ACEI2，并建立了其分离纯化方法。经检测，在0.01mg/mL浓度下，ACEI1的ACE抑制率可达73.61％，ACEI2对其的抑制率为59.82％，所获得的新型ACE抑制肽可有望用于食品、功能性食品、药品等行业中。
Description
技术领域
本发明属于生物医药及食品领域，是酸乳中两种新型ACE抑制肽及其基因工程生产。
背景技术
高血压是危害人类身体健康最常见的心血管疾病，它不仅患病率高，而且常引起其他心血管疾病。由于高血压在早期阶段通常无明显症状，仅少数人有头痛、头晕、心悸、四肢麻木等症状，而晚期则将引起心、脑、肾等器官的病变，因此高血压被称为人类健康无形的杀手，已成为全球范围内的重要公共卫生问题。
血管紧张素转换酶(Angiotension Convening Enzyme，ACE)一种Zn2+依赖型羧二肽酶，ACE在人体内通过肾素-血管紧张素系统(Renin-angiotensin system，RAS)和激肽释放酶-激肽系统(Kallikrein-kinin system，KKS)参与血压调节。ACE是RAS系统的关键酶，在Zn2+存在的条件下，ACE能切下AngI羧基末端的二肽(His-Leu)，使之活化变成血管紧张素Ⅱ(AngⅡ)。AngⅡ是目前研究发现最强的血管收缩物质之一，它作用于血管紧张素受体1，收缩血管平滑肌，同时刺激醛固酮分泌，促进人体肾脏对Na+，K+的重吸收，引起血容量和钠贮量增加，使血压升高。KKS系统中的激肽如缓激肽的主要作用是作用于血管内皮的缓激肽受体，促进一氧化氮等血管扩张因子的释放，促进血压下降。ACE能水解缓激肽，钝化其扩张血管的生物活性，导致血管收缩血压升高。RAS和KKS在血压调节方面是一对相互拮抗的体系，其平衡协调对维持正常血压有重要作用，ACE活力的升高会破坏正常体液中升压和降压体系的平衡，从而导致血压升高，因此抑制ACE的活性对降压有积极影响。
目前，通过ACE的构效关系，已研发并上市了一系列的ACE抑制剂药物。根据它们活性部分化学结构的不同可分为3类：第1类为琉基类，代表药为卡托普利；第2类为磷酸基团类，代表药为福辛普利；第3类为羧基类，代表药为依那普利。ACE抑制剂能抑制心肌、肾脏、血管壁上的ACE活性，除了降压，还具有抑制心肌和血管肥厚的作用，显著改善慢性心力衰竭患者的自主神经活性及自主性平衡，因此ACE抑制剂除作为降压药已被广泛应用之外，目前也被用于治疗心血管系统、泌尿系统、内分泌系统的多种疾病。许多研究证实，长期使用这类药可明显改善心脏病人的心功能，减少多种肾脏病人的尿蛋白，延缓肾功能衰竭的发生，提高病人生活质量，延长病人的生命期限。
随着生活水平的提高，人们己经不仅仅满足于药物的治疗效果，对其安全性和副作用的影响也愈加为人们所重视。而化学合成的ACE抑制剂则往往易产生干咳等不良反应。随着研究工作的开展，人们陆续发现了许多来源于食物蛋白的ACE抑制肽，这些食品源的ACE抑制肽因其天然无毒等优点引起了人们极大的关注。实验表明这些食物蛋白源ACE抑制肽对自发性高血压大鼠及高血压患者具有明显的降压作用，而对正常血压大鼠及人无降压作用。目前，用于制备食品源ACE抑制肽的原料大致分为两类：植物蛋白和动物蛋白。植物来源以大豆蛋白为主；动物来源则以乳蛋白和水产品蛋白为主。
目前已发现的食品源ACE抑制肽均为2-30个氨基酸组成的小肽。但因其分子量较小(3kD以下)，分离纯化困难，难以通过基因工程及微生物发酵方法进行高效生产，而化学合成则成本较高，因此限制了其产业化开发及实际应用。
发明内容
本发明提供一种酸乳中新型ACE抑制肽及其基因工程生产方法，首次从酸乳中分离并鉴定了两个分子量显著大于已报道食品源ACE抑制肽的新型ACE抑制肽ACEI1(18.3kD)和ACEI2(12.2kD)，建立了其酸乳中分离提取生产方法及基因工程表达生产方法。
为达到上述目的，本发明采用以下技术方案：
一种酸乳中新型ACE抑制肽，命名为ACEI1和ACEI2，其分子量分别为18.3kD和12.2kD，ACEI1的氨基酸序列为序列1，ACEI2的序列2所示。
一种如权利要求1所述的酸乳中新型ACE抑制肽的发酵菌株，菌株名称为：HZ1，分类命名为：干酪乳杆(Lactobacillus Casei)，保藏号为CGMCC No.13312，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为：北京市朝阳区北辰西路1号院3号，保藏日期为：2016年11月15日。
一种酸乳中新型ACE抑制肽作为食品原料、功能食品原料、药物原料的应用。
一种含有酸乳中新型ACE抑制肽食品级重组菌作为食品新原料、功能食品原料、药物原料的应用。
一种发酵获得酸乳中新型ACE抑制肽的菌株作为食品原料、功能食品原料、药物原料的应用。
一种能够表达酸乳中新型ACE抑制肽的生物工程菌，构建过程主要包括如下步骤：
步骤1：根据抑制肽ACEI1、ACEI2的氨基酸序列，利用宿主菌密码子偏爱性优化编码基因序列；
步骤2：通过PCR或人工合成获得两种ACE抑制肽基因，将基因插入到表达载体的多克隆位点，构建得到ACEI1及ACEI2重组质粒；
步骤3：将ACEI1、ACEI2重组质粒转化至宿主菌E.coli BL21(DE3)或E.coliRosetta(DE3)中，筛选得到ACEI1、ACEI2重组菌。
与现有技术相比本发明的有益效果和优点是：
1、本发明首次从酸乳中分离并鉴定了两个新型ACE抑制肽ACEI1和ACEI2，其生物学活性高，在0.01mg/mL浓度下，ACEI1的ACE抑制率可达73.61％，ACEI2为59.82％。可为食品、功能食品级药品等行业提供一种优良、安全的降血压活性物质。
2、本发明的两个新型ACE抑制肽分子量分别为12.2kD、18.3kD，显著大于已报道食品源ACE抑制肽(<5kD)，具有分离纯化难度较低、易于微生物发酵及基因工程表达生产等优点，产业化难度较低。
3、本发明建立了两个新型ACE抑制肽从酸乳中直接分离提取的生产方法，可获得高生物学活性及高纯度的ACE抑制肽产品。
4、本发明建立了两个新型ACE抑制肽的基因工程表达生产方法，与直接从酸乳中分离提取相比，更进一步降低了分离纯化难度、缩短了生产工艺，减少了生产成本，适于更好的大规模工业化生产与应用。
5、本发明所述的Lactobacillus Casei HZ1及其发酵乳可直接作为食品新原料、功能食品原料、药物原料加以应用。
6、本发明所述的新型ACEI肽食品级重组菌可直接作为食品新原料、功能食品原料、药物原料加以应用。
7、本发明获得的ACE抑制肽生物学活性高，所建立的酸乳提取纯化方法及基因工程表达生产方法可有效制备高纯度的新型ACE抑制肽ACEI1及ACEI2。其中，与直接从酸乳中分离提取相比，基因工程表达生产方法更进一步降低了分离纯化难度、缩短了生产工艺，减少了生产成本。因此，本发明所述的酸乳及其发酵菌株、两种新型ACE抑制肽及其食品级重组菌可作为优良、安全的降血压活性原料应用于食品、功能食品、药品等产品中，所建立的ACE抑制肽生产方法适于大规模工业化生产。
附图说明
图1PCR产物的琼脂糖凝胶电泳图。
图2为本发明乳酸菌(以Lactobacillus Casei HZ1为例)发酵酸牛乳经分离纯化至RP-HPLC时的色谱图，其中洗脱时间28.2min和24.1min的峰分别为新型ACE抑制肽ACEI1和ACEI2。
图3为本发明两种新型ACE抑制肽基因工程表达的电泳检测图。泳道M：Marker；1、3、5、7：对照宿主菌表达产物；2、4：ACEI1重组菌表达产物；6、8：ACEI2重组菌表达产物。箭头所指为目的产物。
图4A和图4B为本发明两种新型ACE抑制肽ACEI1(图4A图)及ACEI2(图4B图)纯化产物的电泳检测图。
图5为本发明两种ACE抑制肽ACEI1、ACEI2纯化产物的活性分析图，其中卡托普利作为阳性对照品，对照宿主菌产物作为阴性对照品。
具体实施方式
下面通过具体实施例对本发明作进一步详述，以下实施例只是描述性的，不是限定性的。
本发明中HZ1菌株的来源如下：样品来源：样品采集自内蒙古家庭式生产的奶酪。
将采集的奶酪样品混匀，用无菌水对样品进行系列稀释，取1g样品加入10mL无菌水，取稀释后的奶酪0.1mL在MRS固体培养基上进行涂布。培养48h后从分离平板上挑取产生透明圈的菌落进行三区划线纯化，经镜检确认为纯种后，挑取单菌落于MRS液体培养基中培养48h。取菌液与60％甘油按3:1的体积比混合均匀后储存于-80℃备用。
MRS固体培养基的配方及条件为：将10g蛋白胨、10g牛肉膏、5g酵母膏、5g乙酸钠、2g柠檬酸三胺、2g磷酸氢二铵、0.58g七水硫酸镁、0.25g四水硫酸锰、1mL吐温加入到900mL蒸馏水中进行充分溶解与混匀，用pH调节器将pH值调到6.0，然后放入121℃灭菌锅灭菌20min。将20g葡萄糖充分溶解于100mL蒸馏水中，并将其放入115℃灭菌锅中灭菌15min。最后将两者混合即是本实验所用的MRS培养基。
菌株的分子生物学鉴定
储存在-80℃的分离菌株按1％的接菌量接种到MRS液体培养基中于37℃培养箱静置培养12-15h，菌液DNA提取使用TIANGEN细菌提取试剂盒按照其说明书方法进行。使用下列引物27f 5’AGAGTTTGAT CCTGGCTCAG 3’和1492r5’GGTTACCTTGTTACGACTT 3’对该细菌的16S rRNA基因序列进行PCR扩增，扩增结果见图1。PCR反应条件如下：95℃，10min；94℃，1min；55℃，1min；72℃，2min，30个循环；72℃，10min。扩增片段长度为1500bp左右。PCR扩增产物纯化后交由华大基因公司进行分析测定。测序结果在G e n b a n k上进行b l a s t比对，结果显示从家庭式奶酪中分离到的乳酸菌是干酪乳杆菌，命名为LactobacillusCasei HZ1。
筛选得到的菌株名称为：HZ1，分类命名为：干酪乳杆(Lactobacillus Casei)，保藏号为CGMCC No.13312，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为：北京市朝阳区北辰西路1号院3号，保藏日期为：2016年11月15日。
筛选得到上述Lactobacillus Casei HZ1，利用该菌，本发明继续获得了两种新型ACE抑制肽，具体说明如下：
本发明提供酸乳中两种新型ACE抑制肽及其生产方法，所述生产方法包括酸乳中分离提取及基因工程表达生产。所述两种新型ACE抑制肽ACEI1和ACEI2，其分子量分别为18.3kD和12.2kD，氨基酸序列为序列1、序列2所示。
所述两种新型ACE抑制肽来源的酸乳，乳酸菌为干酪乳杆菌Lactobacillus CaseiHZ1，发酵原料为牛乳。
所述两种新型ACE抑制肽从酸乳中分离提取生产方法，主要包括如下步骤：
步骤1：乳酸菌(例如L.Casei HZ1)在MRS等培养基中活化3代后，以3％体积比的接种量转到纯乳(115℃，15min灭菌)中，再活化3代，直到菌种活力完全恢复，作为发酵种子液。将发酵种子液以10％体积比的接种量接入到纯乳中，37℃厌氧培养24h制备得到酸乳；
步骤2：所得发酵酸乳经12000rpm，4℃离心20min后，得到酸乳上清液；
步骤3：酸乳上清液依次经过阴离子交换层析、凝胶层析、反相高效液相(RP-HPLC)分离纯化后，得到ACEI1和ACEI2。
所述的新型ACE抑制肽基因工程表达生产，所用宿主菌及表达质粒为常规基因工程表达系统宿主菌及表达质粒，包括大肠杆菌及食品级基因工程宿主菌(乳酸菌、枯草芽孢杆菌、酵母菌)及其表达质粒等。
所述新型ACE抑制肽重组菌的构建过程包括：
1)根据两种新型ACE抑制肽ACEI1、ACEI2的氨基酸序列(具体序列见序列1、序列2)，利用宿主菌密码子偏爱性优化编码基因序列；
2)通过PCR或人工合成获得两种ACE抑制肽基因，将基因插入到表达载体的多克隆位点，构建得到ACEI1及ACEI2重组质粒；
3)将ACEI1、ACEI2重组质粒转化至宿主菌，筛选得到ACEI1、ACEI2重组菌。
两种新型ACE抑制肽作为食品新原料、功能食品原料、药物原料的应用。
Lactobacillus Casei HZ1及其发酵乳作为食品新原料、功能食品原料、药物原料的应用。
新型ACEI肽食品级重组菌作为食品新原料、功能食品原料、药物原料的应用。
具体操作步骤及条件采用以下实施例来说明。
实施例1
酸乳的发酵制备及新型ACE抑制肽的分离纯化
1、乳酸菌(例如L.Casei HZ1)在MRS等培养基中活化3代后，以3％体积比的接种量转到纯乳(115℃，15min灭菌)中，再活化3代，直到菌种活力完全恢复，作为发酵种子液。将发酵种子液以10％体积比的接种量接入到纯乳中，37℃厌氧培养24h制备得到酸乳；
2、所得发酵酸乳经12000rpm，4℃离心20min后，得到酸乳上清液；
3、酸乳上清液依次经过DEAE弱阴离子交换层析和Sephadex G-15凝胶层析分离后，将经过粗分离所得的ACE抑制肽样品经0.45μm微孔滤膜过滤，取滤液进行反相高效液相(RP-HPLC)分离纯化：(1)色谱柱采用C18色谱柱；(2)流动相：水和乙睛。流动相应过0.45μm膜后备用，使用前超声20min。再应用；(3)具体洗脱条件如下表所示：
实施例2
新型ACE抑制肽的基因工程表达生产
1.新型ACE抑制肽
所用宿主菌及表达质粒为常规基因工程表达系统宿主菌及表达质粒，包括大肠杆菌以及食品级基因工程宿主菌(乳酸菌、芽孢杆菌、酵母菌等)及其表达质粒等，构建过程主要包括如下步骤：
(1)根据两种新型ACE抑制肽ACEI1、ACEI2的氨基酸序列(具体序列见序列1、序列2)，利用宿主菌密码子偏爱性优化编码基因序列，；
(2)通过PCR或人工合成获得两种ACE抑制肽基因，将基因插入到表达载体的多克隆位点，构建得到ACEI1及ACEI2重组质粒；
(3)将ACEI1、ACEI2重组质粒转化至宿主菌E.coli BL21(DE3)或E.coli Rosetta(DE3)中，筛选得到ACEI1、ACEI2重组菌。
(4)通过对诱导表达后的菌体进行超声破碎，来分析重组菌对于目的蛋白ACEI1和ACEI2的表达情况。
(5)通过Ni柱对重组蛋白进行纯化，使用从低到高不同浓度的洗脱缓冲液对重组蛋白进行洗脱，得到ACEI1和ACEI2的纯化产物。
实施例3
产物ACE抑制活性分析
干酪乳杆菌在液体培养基(MRS)中活化3代后，以3％体积比的接种量转到纯牛奶(115℃，15min灭菌)中，活化3代以上，直到菌种活力完全恢复，作为发酵种子液，以10％体积比的接种量接入到纯牛奶中，37℃厌氧培养24h，所得发酵液12000r/min，4℃离心20min，取上清液，用0.22μm针式过滤器过滤，获得清液用于测ACEI活性。
取ACE粗酶液0.15mL→加样品抑制剂(ACEI)0.10mL→37℃水浴中5min→加0.10mL的底物HHL(Hip-His-Leu)，混匀→37℃水浴中孵育60min→立即加入1mol/L的HCl 0.25mL终止反应→静置5min→加乙酸乙酯1.50mL进行萃取，强烈振荡1min，3000r/min离心5min→取酯层0.5mL，在120～130℃下蒸干→加1mol/L NaCl 3mL溶解残渣→于228nm处，以1mol/LNaCl溶液作参比比色。测值记作S；用水代替样品抑制肽，步骤同上，测得A值；用水代替粗酶液和酶解肽液测得C值。ACE抑制率(％)＝(A-S)/(A-C)×100。结果经计算，抑制率高达99.12％。
Claims (4)
1.酸乳中ACE抑制肽，其特征在于：命名为ACEI1和ACEI2，其分子量分别为18.3 kD和12.2 kD，ACEI1的氨基酸序列如SEQ ID NO:1所示，ACEI2的氨基酸序列如SEQ ID NO:2所示。
2.一种如权利要求1所述的酸乳中ACE抑制肽在制备降压药物的应用。
3.一种含有如权利要求1所述的酸乳中ACE抑制肽食品级重组菌在制备降压药物中的应用。
4.一种能够表达权利要求1所述的酸乳中ACE抑制肽的生物工程菌，其特征在于：构建过程主要包括如下步骤：
步骤1：根据抑制肽ACEI1、ACEI2的氨基酸序列，利用宿主菌密码子偏爱性优化编码基因序列；
步骤2：通过PCR或人工合成获得两种ACE抑制肽基因，将基因插入到表达载体的多克隆位点，构建得到ACEI1及ACEI2重组质粒；
步骤3：将ACEI1、ACEI2重组质粒转化至宿主菌E.coliBL21(DE3)或E.coliRosetta(DE3)中，筛选得到ACEI1、ACEI2重组菌。
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