CN113336825B - 一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽及其应用 - Google Patents
一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有α‑葡萄糖苷酶和α‑淀粉酶抑制活性的六肽，所述六肽氨基酸序列为Pro‑Trp‑Leu‑Tyr‑Phe‑Ile。本发明还公开了其在制备降血糖食品、保健品、药品中的应用。本发明从山核桃蛋白中筛选得到了能够有效抑制α‑葡萄糖苷酶活性和α‑淀粉酶活性的六肽PWLYFI，并明确六肽PWLYFI与α‑葡萄糖苷酶和α‑淀粉酶的作用位点，对开发具有降血糖的食品、保健品和药品具有十分重要的意义。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽及其应用。
背景技术
糖尿病(Diabetes Mellitus,DM)是一种以高血糖为特征的慢性代谢性疾病，主要由遗传和环境两类因素共同作用。如果不加以控制，将会引起糖类、脂类和蛋白质等代谢紊乱，继而引发多种并发症，包括糖尿病心血管并发症、糖尿病眼部并发症、糖尿病神经病变等(Díaz Naya&Delgado
2016；Gabler et al.,2021)。目前，糖尿病已经成为人类主要的死亡原因之一(Annamalai,Kosir,&Tek,2017；Khajebishak,Payahoo,Alivand,&Alipour,2019)。
人体血糖主要来源于饮食，其中主要的碳水化合物为链段较大的淀粉和糖分子，不能直接吸收进入血循环，需要经过人体的α-淀粉酶和小肠黏膜上的多种α-葡萄糖苷酶的作用下生成葡萄糖及果糖后才能被吸收进入血液进而提高血糖。因此，可以通过抑制α-葡萄糖苷酶和α-淀粉酶的活性来起到减少葡萄糖的生成，从而起到降低血糖的作用(Jin etal.,2016)。例如阿卡波糖作为从放线菌代谢产物获得的一种多糖类物质，是目前临床上常用的一种口服降糖药(Cheng,Liu,Wu,Donkor,Li,&Ma,2017)。阿卡波糖是一种假性四糖物质，可以与α-葡萄糖苷酶发生竞争性抑制作用，从而延缓葡萄糖的吸收，来达到降低餐后血糖的目的。但长期服用阿卡波糖会伴随有胃肠道痉挛胀气等副作用，并且会不可避免地产生耐药性(Fang et al.,2017)。因此，毒副作用较小且能有效缓解血糖的小分子肽受到越来越多的关注(Chan-Zapata,Sandoval-Castro,&Rubi Segura-Campos,2020)。
目前关于生物活性肽的研究的弊端在于对蛋白质和蛋白酶的选择较为随机，且对于活性肽的分离纯化较为繁琐以及耗时，影响了对于活性肽活性的研究。分子对接(Molecular docking)技术作为一种基于计算机模拟技术，能够将小分子配体与大分子受体模拟结合，通过化学计量学方法来模拟分子的几何匹配和能力匹配，不断优化小分子配体位置、氨基酸侧链等空间骨架来达到大分子受体与小分子配体的空间匹配，从而分析不同配体与受体之间的结合模式以及结合的紧密程度。分子对接技术具有快速、高效的特点，已广泛应用于小分子领域(Xu et al.,2021；Yu et al.,2021)。分子对接的广泛利用可以清晰地了解活性肽和受体蛋白的对接位点及二者之间存在的相互作用力，从而分辨出肽段的生物活性和有效性，有助于我们快速地筛选出具有生物活性的肽段。
本发明旨在提供一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽，并明确该六肽与α-葡萄糖苷酶和α-淀粉酶的作用位点，对开发具有降血糖的食品、保健品和药品具有十分重要的意义。
Díaz Naya,L.,&Delgado
E.(2016).Diabetes mellitus.Criteriosdiagnósticos y clasificación.Epidemiología.Etiopatogenia.Evaluación inicialdel paciente con diabetes.Medicine-Programa de Formación Médica ContinuadaAcreditado,12(17),935-946.
Gabler,M.,Picker,N.,Geier,S.,Ley,L.,Aberle,J.,Lehrke,M.,.Wilke,T.(2021).Guideline Adherence and Associated Outcomes in the Treatment of Type2Diabetes Mellitus Patients With an Incident Cardiovascular Comorbidity:AnAnalysis Based on a Large German Claims Dataset.Diabetes Therapy.
Annamalai,A.,Kosir,U.,&Tek,C.(2017).Prevalence of obesity anddiabetes in patients with schizophrenia.World Journal of Diabetes,8(8),390-396.
Khajebishak,Y.,Payahoo,L.,Alivand,M.,&Alipour,B.(2019).Punicic acid:Apotential compound of pomegranate seed oil in Type 2diabetes mellitusmanagement.Journal of Cellular Physiology,234(3),2112-2120.
Jin,J.,Ma,H.,Wang,W.,Luo,M.,Wang,B.,Qu,W.,...Li,Y.(2016).Effects andmechanism of ultrasound pretreatment on rapeseed protein enzymolysis.Journalof the Science of Food and Agriculture,96(4),1159-1166.
Cheng,Y.,Liu,Y.,Wu,J.,Donkor,P.O.,Li,T.,&Ma,H.(2017).Improving theenzymolysis efficiency of potato protein by simultaneous dual-frequencyenergy-gathered ultrasound pretreatment:Thermodynamics andkinetics.Ultrasonics Sonochemistry,37,351-359.
Fang,Y.,Wang,S.,Wu,J.,Zhang,L.,Wang,Z.,Gan,L.,...Hou,J.(2017).Thekinetics and mechanism of alpha-glucosidase inhibition by F5-SP,a novelcompound derived from sericin peptides.Food Funct,8(1),323-332.
Chan-Zapata,I.,Sandoval-Castro,C.,&Rubi Segura-Campos,M.(2020).Proteins and peptides from vegetable food sources as therapeutic adjuvantsfor the type 2diabetes mellitus.Critical Reviews in Food Science andNutrition.
Xu,Z.,Wu,C.,Sun-Waterhouse,D.,Zhao,T.,Waterhouse,G.I.N.,Zhao,M.,&Su,G.(2021).Identification of post-digestion angiotensin-I converting enzyme(ACE)inhibitory peptides from soybean protein Isolate:Their productionconditions and in silico molecular docking with ACE.Food Chemistry,345.
Yu,Z.,Kang,L.,Zhao,W.,Wu,S.,Ding,L.,Zheng,F.,...Li,J.(2021).Identification of novel umami peptides from myosin via homology modeling andmolecular docking.Food Chemistry,344.
发明内容
本发明的目的是提供一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽及其应用，以解决现有技术的不足。
本发明采用以下技术方案：
本发明第一方面提供了一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽，所述六肽氨基酸序列为Pro-Trp-Leu-Tyr-Phe-Ile，缩写为PWLYFI。
本发明第二方面提供了上述六肽在制备降血糖食品、保健品、药品中的应用。
本发明的有益效果：
本发明从山核桃蛋白中筛选得到了能够有效抑制α-葡萄糖苷酶活性和α-淀粉酶活性的六肽PWLYFI，并明确六肽PWLYFI与α-葡萄糖苷酶和α-淀粉酶的作用位点，对开发具有降血糖的食品、保健品和药品具有十分重要的意义。
附图说明
图1是PWLYFI与α-葡萄糖苷酶的对接结果3D图。
图2是PWLYFI与α-淀粉酶的对接结果3D图。
图3是PWLYFI的HPLC图。
图4是PWLYFI的质谱图。
图5是PWLYFI对α-葡萄糖苷酶的抑制结果图。
图6是PWLYFI对α-淀粉酶的抑制结果图。
具体实施方式
下面结合实施例和附图对本发明做更进一步地解释。下列实施例仅用于说明本发明，但并不用来限定本发明的实施范围。
实施例1：具有α-葡萄糖苷酶和α-淀粉酶抑制作用的活性肽的筛选
首先将山核桃去壳后通过液压榨油机冷榨脱脂，条件为室温，50Mpa，40min。之后将得到的山核桃饼粕粉碎，用石油醚以1:5(g/mL)的料液比于室温下提取1h后进行抽滤，共提取两次后收集滤渣，50℃下干燥至含水量低于4wt％后粉碎过60目筛，于4℃下冷藏备用。
采用碱提酸沉法提取蛋白：用蒸馏水将上述得到的山核桃脱脂粉按照1：17(g/mL)的料液比制成蛋白质溶液，用1.0mol/L NaOH溶液调节pH至10.0，于50℃水浴3h后，7000r/min 4℃离心20min，取上清液，用1.0mol/L HCl溶液调节pH至4.0，静置2h后，7000r/min 4℃离心20min，取沉淀，蒸馏水洗至中性后-80℃冷冻干燥24h后得山核桃蛋白，于-20℃保存备用。
按照2％w/v的底物浓度称取一定量的山核桃蛋白加蒸馏水制成蛋白质溶液，于90℃水浴锅中变性10min后冷却至室温，用1.0mol/L NaOH溶液调节至pH7.7，加入3900U/g(以山核桃蛋白的质量为基准)的复合蛋白酶(所用复合蛋白酶为上海源叶生物科技有限公司所售的复合蛋白酶，货号为S10155，主要包括枯草芽孢杆菌产生的内切蛋白酶)，于55℃条件下酶解2h。酶解结束后于95℃水浴锅中灭酶10min后冷却至室温，7000r/min 4℃离心20min，取上清液，过0.22μm滤膜，再利用3kDa MWCO超滤膜进行超滤。将滤过液收集，-80℃冷冻干燥24h，于-20℃保存备用。之后利用Nano LC-MS/MS技术获得该组分序列信息。
使用peptide property calculator、PeptideRanker和ToxinPred分别对所有肽进行水溶性、肽生物活性评分和毒性测定。筛选得到水溶性好、肽生物活性评分高于0.8、无毒的31条肽，分别为PWLYFI、HAFFPL、HGAFGLIGFML、VPWKMF、WIFWVGP、FPPGKMP、VLISLFFF、IWDPHFG、FLKDPF、IDNIFRF、SPCIIWIPN、KPFAWARRAL、GLPPLAGF、QPFMRWRD、FIPNDFP、WLKNLFL、QFPEW、KPPFFS、RIWRP、INGWLR、LFKWDP、PWGMFLGSVRR、SVPGAMF、KAGYPLGGL、ADDIYMGPRC、ALGHYVHIWW、FPECIL、VLPPIFY、SYDMLGIFY、GYASMITYMLF、FQGPPHG。
从RCSB PDB数据库(http://www.rcsb.org/)下载α-葡萄糖苷酶的晶体结构(3WY1)和α-淀粉酶的晶体结构(1BAG)，并将其作为蛋白靶标，通过Autodock Vina程序进行分子对接来筛选能与α-葡萄糖苷酶和α-淀粉酶紧密结合的多肽，并明确活性位点。以结合最低结合能、形成氢键的数目及作用的关键氨基酸为指标，筛选得到具有潜在α-葡萄糖苷酶抑制活性和α-淀粉酶抑制活性的六肽PWLYFI，氨基酸序列如SEQ ID NO.1所示，为Pro-Trp-Leu-Tyr-Phe-Ile，缩写为PWLYFI。结果表明，PWLYFI与α-葡萄糖苷酶对接的结合能为-10.1Kcal/mol，与α-葡萄糖苷酶的主要氨基酸残基即ASP-40、GLN-439、ARG-450、HIS-515和ALA-529结合(图1)；与α-淀粉酶对接的结合能为-9.2Kcal/mol，可以与α-淀粉酶的主要氨基酸残基即ALA-177、GLN-208和ASP-275结合(图2)。
本发明提供的六肽可源自山核桃饼粕水解物，也可通过固相合成得到。实施例2和实施例3所用到的PWLYFI为固相合成所得(南京金斯瑞生物科技有限公司)，HPLC图和质谱图分别如图3和图4所示。
实施例2：体外α-葡萄糖苷酶抑制活性鉴定
采用分光光度法测定PWLYFI的α-葡萄糖苷酶抑制活性。首先用0.2M pH 6.8的磷酸缓冲盐溶液(PBS)配置0.1U/mLα-葡萄糖苷酶溶液和2.5mM pNPG溶液。将2.8mL PBS、0.5mLα-葡萄糖苷酶溶液和0.5mL 1mmol/L多肽(PWLYFI)溶液混合，于37℃下孵育10min。然后加入0.5mL pNPG溶液，于37℃下孵育40min。最后加入2mL 0.2M Na2CO3溶液终止反应，于405nm下测定吸光度。
采用下列公式计算六肽的抑制率：
抑制率(％)＝(1-(A1-A2)/A3)×100％
其中，A1为实验组，A2为样品空白组，A3为对照组。
添加0.5mL多肽溶液的为实验组A1，以0.5mL PBS替代α-葡萄糖苷酶溶液的为样品空白组A2，以0.5mL PBS替代多肽溶液的为对照组A3。
结果表明(图5)，PWLYFI可以抑制α-葡萄糖苷酶活性，在0.5mg/mL、1.0mg/mL、1.5mg/mL和2.0mg/mL时抑制率分别达到35.53±3.56％、57.17±6.32％、52.84±2.69％和55.15±3.16％。
实施例3：体外α-淀粉酶抑制活性鉴定
采用分光光度法测定PWLYFI的α-淀粉酶抑制活性。首先配置3,5-二硝基水杨酸(DNS)试剂，取6.3g DNS和262mL 2M NaOH溶液加入到500mL含185g四水合酒石酸钾钠的水溶液中，再加入5g苯酚和5g亚硫酸钠(Na2SO3)，超纯水定容至1L，于棕色瓶室温保存。用0.2MpH 6.8的磷酸缓冲盐溶液(PBS)配置0.1U/mLα-淀粉酶溶液。将淀粉溶解于PBS中并煮沸，冷却后用PBS定容以配置2％w/v淀粉溶液。取0.5mL 1mmol/L多肽(PWLYFI)溶液与0.5mLα-淀粉酶溶液于25mL具塞比色管中，于37℃下反应30min。之后加入1mL 2％w/v淀粉溶液，于37℃下反应15min，再加入2mL DNS试剂终止反应，最后在沸水浴下加热5min，流水(自来水)冷却至室温后PBS定容至刻度(25mL)，于540nm下测定吸光度。
采用下列公式计算六肽的抑制率：
抑制率(％)＝(1-(A1-A2)/A3)×100％
其中，A1为实验组，A2为样品空白组，A3为对照组。
添加0.5mL多肽溶液的为实验组A1，以0.5mL PBS替代α-淀粉酶溶液的为样品空白组A2，以0.5mL PBS替代多肽溶液的为对照组A3。
结果表明(图6)，PWLYFI可以抑制α-淀粉酶活性，在0.5mg/mL、1.0mg/mL、1.5mg/mL、2.0mg/mL和2.5mg/mL时抑制率分别达到21.46±4.54％、51.71±2.76％、62.46±3.46％、67.12±4.65％和69.14±2.41％。
以上所述的实施例对本发明的技术方案进行了详细说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围的前提下，还可以做出各种变化和变型，这些变化和变型也应视为本发明的保护范围。
序列表
<110> 浙江农林大学
<120> 一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> PWLYFI(六肽)
<400> 1
Pro Trp Leu Tyr Phe Ile
1 5
Claims (2)
1.一种具有α-葡萄糖苷酶和α-淀粉酶抑制活性的六肽，其特征在于，所述六肽氨基酸序列为Pro-Trp-Leu-Tyr-Phe-Ile。
2.权利要求1所述的六肽在制备降血糖药品中的应用。
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