CN116478237A - 一种胰脂肪酶抑制多肽组合及其制备方法、应用 - Google Patents
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Abstract
本发明公开一种胰脂肪酶抑制多肽组合的制备方法，包括以下步骤：1）取鲣鱼红肉加入双蒸水匀浆，待用；2）取步骤1）得到的鲣鱼红肉匀浆加入碱性蛋白酶进行酶解处理；3）将步骤2）得到的酶解液冷冻干燥；4）将步骤3）得到的酶解液冷冻粉进行多肽de novo从头测序；5）根据多肽序列结果，用胆固醇酯酶分子对接，得到胰脂肪酶抑制多肽组合（FPLPMPFLDL，QAPLDLPEPFL和/或FLPMPL）。本发明所提供的鲣鱼胰脂肪酶抑制多肽组合（FPLPMPFLDL，QAPLDLPEPFL和/或FLPMPL）对胰脂肪酶有抑制作用，并且具有显著的降脂活性。
Description
技术领域
本发明属于生物技术领域，具体涉及一种胰脂肪酶抑制多肽组合及其制备方法、应用。
背景技术
鲣鱼属于金枪鱼的一个种类，鲣鱼也被称为小金枪鱼，具有高蛋白、低脂肪等特点，富含二十二碳六烯酸(DHA)、二十碳五烯酸(EPA)等具有生物活性的多不饱和脂肪酸，同时，甲硫氨酸、牛磺酸、矿物质和维生素的含量丰富，是国际营养协会推荐的绿色无污染健康美食。金枪鱼产品以生鱼片和鱼罐头为主，备受消费者青睐，但加工过程会产生较多副产物，例如鱼皮、内脏、碎肉、鱼骨等，占金枪鱼重量的50%-70%。目前，副产物主要被加工为鱼饵或动物饲料，商业价值较低。鲣鱼作为一种捕捞量大且价格低廉的金枪鱼具有强大的市场潜力。但由于其肉质发酸，口感不佳，且暗色肉比例大，腥味重，故一直未被很好的开发利用，一般只作为罐头或者木鱼精调味品进行销售，相关产品的研究甚少，经济效益低。如何有效利用鲣鱼暗色肉开发高附加值产品已引起水产加工企业的重视。
近年来，大量研究表明食物蛋白源生物活性肽是调节机体代谢、促进人体健康的潜在功效因子。生物活性肽通常由2-20（也有观点认为是2-50）个氨基酸组成，这些序列往往因被包埋在母蛋白序列中而为表现出生物活性，经发酵、酶解以及胃肠道消化等手段水解释放后，通常表现出更强的活性。与蛋白质相比，肽具有低分子量、易被吸收、高稳定性等特点。科研人员近年来针对鲣鱼红肉酶解肽开展了一系列的研究工作，李淑凡等制备了金枪鱼红肉酶解肽，该酶解肽对小鼠抗疲劳和调节肠道菌群发挥积极作用，经分析，酶解液中相对含量前十的多肽序列含有4-11个氨基酸（205-1199Da），序列分别为：KEFT，EESAS，RYDD，VEKE，TIRM，FPRM，PVALSCHC，MLGGFGNW，MIGGFGNW，YRDFYYKT和PPCQLINQTVS。蒲月华等研究发现长鳍金枪鱼头多肽TIP3C（200-800 Da）对4种自由基均具有较强的清除作用，且清除率都随着质量浓度的增大而显著增加，剂量效应关系明显。陈悦等制备小分子金枪鱼多肽能通过增加小鼠脑组织SOD和GSH-Px的含量，降低MDA的含量，上调记忆力相关基因的表达量来增强小鼠的记忆力，增加小鼠皮肤弹性；及改善小鼠睡眠质量。孙婷婷等提取金枪鱼胰脏酶解肽可以改善db/db糖尿病小鼠的体质量、血清胰岛素、糖化血红蛋白、血脂水平，其作用效应可能与其下调醛固酮合成相关基因CYP11B1的表达水平有关。Xing-Wei Xiang等酶解鲣鱼获得<1 kDa的多肽能够改善小鼠结肠炎，主要通过提高SOD和GSH-Px及降低炎症因子LPS，IL-6和TNF-α的表达。H Zhong等从鲣鱼鱼肉的酶解液中分离纯化得到五肽(ACECD)，分子量范围为600-1000 Da，具有抗氧化与抑制黄嘌呤氧化酶活性的作用。
发明内容
针对现有技术存在的问题，本发明的目的在于设计提供一种胰脂肪酶抑制多肽组合及其制备方法、应用的技术方案。
本发明具体采用以下技术方案实现：
本发明第一方面提供了一种胰脂肪酶抑制多肽组合，其包括如SEQ ID NO.1所述的多肽、SEQ ID NO.2所述的多肽和/或SEQ ID NO.3所述的多肽。
本发明第二方面提供了上述一种胰脂肪酶抑制多肽组合作为胰脂肪酶抑制剂的应用。
本发明第三方面提供了上述一种胰脂肪酶抑制多肽组合在制备降脂药物中的应用。
本发明第四方面提供了上述胰脂肪酶抑制多肽组合的制备方法，其特征在于包括以下步骤：
1）取鲣鱼红肉加入双蒸水匀浆，待用；
2）取步骤1）得到的鲣鱼红肉匀浆加入碱性蛋白酶进行酶解处理；
3）将步骤2）得到的酶解液冷冻干燥成粉末；
4）将步骤3）得到的酶解液冷冻粉进行多肽de novo从头测序；
5）根据多肽序列结果，用胰脂肪酶分子对接，得到胰脂肪酶抑制多肽组合。
进一步，所述步骤2）中酶解条件为：加酶量5000U/g，pH 8，料液比1：5，酶解温度45℃，酶解时间 10h。
与现有技术相比，本发明所提供的鲣鱼胰脂肪酶抑制多肽组合（FPLPMPFLDL，QAPLDLPEPFL和/或FLPMPL）对胰脂肪酶有抑制作用，并且具有显著的降脂活性。
附图说明
图1为本发明胰脂肪酶抑制多肽组合的筛选的流程图。
具体实施方式
以下结合实施例来进一步说明本发明。
实施例1：胰脂肪酶抑制多肽组合的制备
1）取鲣鱼红肉250g加入150ml双蒸水匀浆，待用；
2）取步骤1）得到的鲣鱼红肉匀浆加入碱性蛋白酶进行酶解处理，酶解条件为：加酶量5000U/g，pH 8，料液比1：5，酶解温度45℃，酶解时间 10h；酶解结束，沸水水浴灭酶15min，冷却后，10000 r/min离心30 min，收集上清液—鲣鱼蛋白酶解液；
3）将步骤2）得到的鲣鱼蛋白酶解液冷冻干燥成粉末；
4）将步骤3）得到的鲣鱼蛋白酶解液冷冻粉进行多肽de novo从头测序并对多肽序列进行分析；
5）根据多肽序列结果，用胰脂肪酶分子对接，得到胰脂肪酶抑制多肽组合（FPLPMPFLDL，QAPLDLPEPFL和/或FLPMPL）。
实施例2：胰脂肪酶抑制多肽组合的筛选
如图1所示，具体的筛选步骤包括：
（1）在筛选的这一阶段，仅选择平均局部置信度（ALC）值至少为60%的多肽序列，因为该值给出了特定氨基酸在序列中准确呈现的高度置信度，建立了包含48684条序列的从头预测肽库。
（2）基于N-to-1神经网络的PeptideRanker服务器从肽库中返回预测的生物活性概率报告，超过阈值80%的1984条多肽被标记为具有潜在生物活性。
（3）在探索可能表现出的潜在生物活性之前，需要预测多肽毒性性质对生物造成的影响，ToxinPred服务器用于预测给定肽的毒性，以帮助在没有开发限制的情况下识别特定肽，SVM得分在0以下被认为无毒，共1939条肽。
（4）1939条无毒肽进一步通过Pepsite2与胰脂肪酶（PDB ID：1ETH）对接，结合的显著性P值小于0.05被认为具有一定抑制潜力；由于Pepsite2的最大输入是10肽，高于10个氨基酸的肽被折中输入，计算平均P值；1939条肽中，1808条肽能与胰脂肪酶1AQL发生显著相互作用，肽库中能与靶酶均产生显著性结合的交集多肽，共1808条肽进一步进行虚拟筛选。
（5）LeDOCK和AutoDock Vina 1.1.2用于虚拟筛选和相互验证，以确定有希望的肽片段。在上述策略的虚拟筛选中，使用以下坐标定义胰脂肪酶（PDB ID： 1ETH）的活性位点：对于LeDock，Xmin= 30，Xmax= 78，Ymin= 22.4，Ymax= 70.4，Zmin= 97.8和Zmax= 145.8，对于Vina，Center_X= 54.0，Center_Y= 46.4，Center_Z= 121.8，Size_X= 48，Size Y= 48和Size_Z=48；以上活性位点区域包含了催化三元组（即SER153、D177和HIS264）和底物结合位点（即F78、I79、H152、F216、W253和R257）。使用X-Score 1.3软件对结合能最低的靶酶-肽复合结构进行共识化评分，结果显示为-logKd的解离常数，μM亲和力即-logKd = 6.0的肽片段被保留；在560条肽中虚拟筛选分析之后，有希望的肽片段进一步与靶酶对接，以证明可能的结合模式。
（6）全局采样服务器HPEPDOCK用于生成100个构象，并由对接评分进行相对排名。利用UCSF Chimera1.15对靶酶活性位点上有希望的肽的结合位置进行可视化和注释，LigPlot+ v.2.2.4列出了靶酶-肽对接的非键合相互作用，使用HawkDock服务器的MM/GBSA模块对绑定结构进行MM/GBSA绑定自由能计算，最终筛选得到：鲣鱼胰脂肪酶抑制多肽组合（FPLPMPFLDL，QAPLDLPEPFL和/或FLPMPL）。
实施例3：胰脂肪酶抑制多肽组合的功能验证
胰脂肪酶抑制率试验：胰脂肪酶能从对硝基苯酚酯中分解出对硝基苯酚，其浓度与 405 nm 波长下吸光度成正比，因此可以测定在一定底物浓度和加酶量条件下，不同浓度多肽对胰脂肪酶活性的抑制效果。
具体验证步骤包括：
1）将胰脂肪酶用缓冲液溶解成 50 mg/mL 酶液，12000 r/min 离心 5 min 取上清酶液备用；
2）底物缓冲液：将对硝基苯酚棕榈酸酯溶解在异丙醇中配成50 mM的溶液再用蒸馏水稀释至10 mM；
3）取 20μL 酶液，15μL底物，样品组体积为 20μL，用 Tris 缓冲液（pH7.4，含 150mM Na Cl，1.3 mM CaCl2）定容至 200μL，37℃反应 2h 后测 405 nm处的吸光度，并根据以下公式计算抑制率：
胰脂肪酶=（对照组吸光度值-样品吸光值）/对照组吸光度值，如表1为FPLPMPFLDL，QAPLDLPEPFL和FLPMPL三条肽取不同浓度时对胰脂肪酶活性抑制率。
表1：
| 浓度（mg/mL）肽序列 | 0.1 | 0.5 | 1.0 | 2.5 |
| FPLPMPFLDL | 25.13%±1.6 | 55.43%±2.6 | 57.68%±3.9 | 69.24%±5.3 |
| QAPLDLPEPFL | 28.26%±1.26 | 32.46%±3.2 | 49.78%±3.1 | 65.91%±4.2 |
| FLPMPL | 38.46%±5.26 | 44.56%±4.3 | 58.65%±2.1 | 73.56%±3.2 |
Claims (5)
1.一种胰脂肪酶抑制多肽组合，其包括如SEQ ID NO.1所述的多肽、SEQ ID NO.2所述的多肽以和/或SEQ ID NO.3所述的多肽。
2.如权利要求1所述的一种胰脂肪酶抑制多肽组合作为胰脂肪酶抑制剂的应用。
3.如权利要求1所述的一种胰脂肪酶抑制多肽组合在制备降脂药物中的应用。
4.如权利要求1所述的胰脂肪酶抑制多肽组合的制备方法，其特征在于包括以下步骤：
1）取鲣鱼红肉加入双蒸水匀浆，待用；
2）取步骤1）得到的鲣鱼红肉匀浆加入碱性蛋白酶进行酶解处理；
3）将步骤2）得到的酶解液冷冻干燥成粉末；
4）将步骤3）得到的酶解液冷冻粉进行多肽de novo从头测序；
5）根据多肽序列结果，用胰脂肪酶分子对接，得到胰脂肪酶抑制多肽组合。
5.如权利要求4所述的制备方法，其特征在于所述步骤2）中酶解条件为：加酶量5000U/g，pH 8，料液比1：5，酶解温度45℃，酶解时间 10h。
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| CN114621336A (zh) * | 2020-12-14 | 2022-06-14 | 西北农林科技大学 | 一种酪蛋白源胰脂肪酶抑制肽及其制备方法 |
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| US20060013769A1 (en) * | 2004-07-16 | 2006-01-19 | Stephen Carter | Transdermal drug delivery formulations and method of determining optimal amounts of vasodilators therein |
| CN114621336A (zh) * | 2020-12-14 | 2022-06-14 | 西北农林科技大学 | 一种酪蛋白源胰脂肪酶抑制肽及其制备方法 |
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