CN114716523B - 具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽 - Google Patents
具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽 Download PDFInfo
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Abstract
具有α‑葡萄糖苷酶抑制活性的小米醇溶蛋白肽。本申请提供了抑制α‑葡萄糖苷酶的熟小米醇溶蛋白肽及其应用和筛选方法,其序列选自SEQ ID NO.1‑3。本申请的肽能够有效抑制α‑葡萄糖苷酶,且安全无毒无副作用,因此作为功能成分用于食品、保健品以及降糖药品中,具有良好的潜力和应用前景。
Description
技术领域
本申请属于蛋白领域,具体地,本申请提供了具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽及其应用和筛选方法。
背景技术
糖尿病是一种以胰岛素激素功能障碍导致的高血糖为特征的代谢性疾病,近年来其患病率呈现快速增长的趋势。其中,2型糖尿病(非胰岛素依赖型)在成人中最为常见,约占所有糖尿病的90%。因此血糖水平的控制对糖尿病患者至关重要;餐后血糖较高可能会增2型糖尿病患病率,进而引发一系列的并发症,如心血管疾病、肾病等。此外,高血糖还会增加活性氧的产生,导致氧化性组织损伤。血糖的主要来源是食物中的碳水化合物,其经过α-葡萄糖苷酶催化水解,生成单糖被吸收。催化水解碳水化合物的α-葡萄糖苷酶主要分布于小肠黏膜刷状缘上。因此,α-葡萄糖苷酶是调控餐后血糖药物的作用靶酶。阿卡波糖、米格列醇和伏格列波糖是目前较多使用的降糖药物,其能够通过竞争性地与α-葡萄糖苷酶结合,抑制该酶的活性,从而减慢糖类水解为葡萄糖的速度,同时减缓小肠对葡萄糖等碳水化合物的消化吸收,达到降低餐后血糖的作用。然而据报道,阿卡波糖和其类似药物会产生一些不良副作用,例如胀气、腹泻、腹部和肝脏疾病。因此,寻找新的具有轻微或没有不良影响的α-葡萄糖苷酶抑制剂对于糖尿病治疗非常重要。
近年来,食物蛋白质作为功能肽的主要来源被广泛研究。许多食源性肽已被证明具有抗菌特性、降血压作用、降低胆固醇、抗血栓形成和抗氧化活性等。小米中的醇溶蛋白能够发挥较好的降糖功效。目前已从小米蛋白中分离出了抗氧化、抗炎以及脂肪酶抑制肽,尚未有关于小米蛋白降糖肽的相关研究。小米醇溶蛋白在通过灌胃被给予糖尿病小鼠后,经过胃肠道消化被水解为氨基酸和肽,进入血液循环发挥包括降糖等生理作用。因此,小米醇溶蛋白水解得到的小分子肽可以作为潜在的功能性降糖物质。
发明内容
针对以上问题,一方面,本申请提供抑具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽,其特征在于,所述肽的序列选自SEQ ID NO.1-3。
进一步地,所述肽的序列为SEQ ID NO.1或SEQ ID NO.2。
另一方面,本申请提供了组合物,其特征在于,所述组合物包含上述肽以及药学、食品或保健品上可接受的辅料。
另一方面,本申请提供了上述肽或者组合物在制备α-葡萄糖苷酶抑制剂中的用途。
另一方面,本申请提供了上述肽或者组合物在制备治疗糖尿病的药物中的应用。
另一方面,本申请提供了上述肽或者组合物在制备适用于糖尿病人群的降糖食品或保健品中的应用。
进一步地,所述糖尿病为2型糖尿病。
另一方面,本申请提供了筛选上述肽的方法,其特征在于,所述方法包括:
(1)体外模拟消化:使用酶法对熟小米醇溶蛋白进行水解以得到蛋白水解物;
(2)筛选:对蛋白水解物进行超滤处理;通过质谱得到超滤级分中置信度较高的肽段序列;采用殷赋云平台对得到的肽段序列进行虚拟筛选,根据肽段的格点打分以及内部排斥能;筛选得到分别与α-葡萄糖苷酶对接效果较好的肽段;
(3)活性位点分析及体外功能预测:对筛选得到的肽序列进行水溶性、不稳定性、等电点、半衰期和ADMET性质的预测;使用殷赋云平台对筛选得到的肽序列与α-葡萄糖苷酶进行对接,确定其与α-葡萄糖苷酶作用的关键氨基酸及相互作用力。
进一步地,所述水解使用胃蛋白酶和胰酶。
进一步地,所述超滤级分为<3kDa的级分。
有益效果:
本次首次从小米醇溶蛋白中筛选得到了能够有效抑制α-葡萄糖苷酶活性的三条未经报道的小肽WFQHQ、YWTPR和FMLPQ,并明确了小肽WFQHQ、YWTPR和FMLPQ的结构;同时WFQHQ、YWTPR和FMLPQ具有安全无毒副作用和良好的人体肠道吸收特性等优点,因此作为功能成分用于食品、保健品以及降糖药品中,具有良好的潜力和应用前景。
附图说明
图1为WFQHQ与α-葡萄糖苷酶的对接结果的3d图;
图2为YWTPR与α-葡萄糖苷酶的对接结果的3d图;
图3为FMLPQ与α-葡萄糖苷酶的对接结果的3d图;
图4为WFQHQ、YWTPR和FMLPQ的α-葡萄糖苷酶抑制率图。
具体实施方式
实施例1小米醇溶蛋白的提取
首先采用盐析法提取小米醇溶蛋白。取一定量的脱脂小米粉,加入70%乙醇(v/v),料液比为1:7,在50℃条件下水浴振荡2小时,反应结束后在7000rpm下离心20min并收集上清液,加入0~4℃的冷盐水稀释至原体积的3倍,缓慢加入并搅拌,使得最终溶液中氯化钠浓度为0.3%,在0-4℃条件下静置1d使蛋白沉淀。然后在7000rpm下离心20min并收集沉淀,加入超纯水与沉淀混合为均匀的悬浊液并透析24h。透析结束后对透析液进行冷冻干燥即可得到小米醇溶蛋白,蛋白纯度为90%
实施例2小米醇溶蛋白的体外模拟消化
将小米蛋白样品与蒸馏水以1:5的比例混合,调节pH至3,加入2000U/mL的胃蛋白酶,反应2h后调节pH到7,再加入胰酶使得消化液中胰酶活力为100U胰蛋白酶/mL消化液,继续反应2h,反应结束后沸水浴灭酶10min以终止反应。最后在7000rpm下离心10min收集沉淀,得到蛋白消化后的水解产物。
实施例3小米醇溶蛋白体外模拟消化产物的超滤
首先用超纯水预清洗Ultra-15离心过滤器,洗后甩干。然后选取分离分子量为3kDa的离心过滤器,向过滤器内加入不超过15mL的样本,将盖好盖子的过滤装置放入离心转子中,转速为5000×g,旋转约30min。离心结束后,取下盖子和过滤器,将离心管中的液体收集并冻干,即为小于3kDa的蛋白水解物超滤样本。
实施例4小米醇溶蛋白体外模拟消化产物的质谱测序及筛选
采用电喷雾-组合型离子阱Orbitrap质谱仪对小于3kDa的水解物组分进行质谱测序,采用De novo的方法进行多肽序列解析,得到所有置信度较高的肽序列。然后基于格点打分小于-70kcal/mol和内部排斥能小于20kcal/mol对测序得到的肽段进行筛选,筛选结果如表1所示。利用PeptideRanker在线系统(http://bioware.ucd.ie/~compass/biowareweb/Server_pages/peptideranker.Php),对表1肽段的生物活性进行预测,筛选得到了生物活性大于0.5的肽段WFQHQ、YWTPR和FMLPQ,表明以上肽段可能具有生物活性。
表1虚拟筛选中与α-葡萄糖苷酶对接效果较好的肽段
实施例5小米醇溶蛋白中筛选得到降糖肽的分子对接及体外功能预测
继续使用殷赋云平台中的Dock 6.9软件进一步进行精确分子对接分析,找出WFQHQ、YWTPR和FMLPQ与酶作用的活性位点。如图1所示,WFQHQ与α-葡萄糖苷酶的残基Trp481、Ala 555、Trp 516、Trp 376、Leu 677、Leu 678和Leu 650形成疏水相互作用,与Gly651和Ser 676形成氢键,与Asp 282和Asp 616形成盐桥,与Phe 649形成π-π堆积。如图2所示,YWTPR与α-葡萄糖苷酶的残基Phe 649、Trp 376、Leu 677和Leu 678形成疏水相互作用,与Asp 616和Gly 651形成氢键,与Arg 600和His 674形成盐桥。如图3所示,FMLPQ与α-葡萄糖苷酶的残基Trp 376、Ala 555和Asp 282形成疏水相互作用,与Asp 282形成盐桥,与Met519、Asp 404和Pro 287形成氢键,与Phe 649形成π-π堆积,与Arg 281形成π-阳离子相互作用。WFQHQ、YWTPR和FMLPQ的稳定性通过EXPASY平台(http://web.expasy.org/protparam/)评估。利用admetSAR预测WFQHQ、YWTPR和FMLPQ的ADMET(http://lmmd.ecust.edu.cn/admetsar1/predict/)性质,主要包括人体肠道吸收(Human intestinal absorption,HIA)和急性口服毒性(Acute Oral Toxicity),结果如表2所示。可以看到,WFQHQ和YWTPR两条肽段均具有较好的体外稳定性、生物活性、人体肠道吸收和低毒性。其中WFQHQ和YWTPR还具有较好的亲水性,且半衰期也较长。
表2肽段WFQHQ、YWTPR和FMLPQ的体外功能预测分析
实施例6小米醇溶蛋白中筛选得到肽段的体外α-葡萄糖苷酶抑制活性测定
首先配置浓度为1.505mg/mL的底物PNPG溶液(5mmol/L,溶于pH=6.8的0.1mol/L磷酸缓冲液),然后是浓度为0.2mol/L的Na2CO3溶液,最后是α-葡萄糖苷酶溶液(0.8U/ml,溶于pH=6.8的0.1mol/L磷酸缓冲液)。溶液配置结束后,设置以下两组进行测定:
按照上表添加后,37℃下水浴振荡10min,然后加入100μL酶液,继续在37℃下水浴振荡10min,最后加入50μL Na2CO3溶液,并在405nm处测定吸光度值。α-葡萄糖苷酶抑制率的计算公式如下:
结果如图4所示,可以看到WFQHQ和YWTPR的α-葡萄糖苷酶抑制率显著高于FMLPQ,分别为34.42%和36.67%,FMLPQ的α-葡萄糖苷酶抑制率最低,为21.66%。
序列表
<110> 中国农业大学
<120> 具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
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Claims (6)
1.具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽,其特征在于,所述肽的序列为SEQID NO.1或SEQ ID NO.2。
2.组合物,其特征在于,所述组合物包含根据权利要求1所述的肽以及药学、食品或保健品上可接受的辅料。
3.根据权利要求1所述的肽在制备α-葡萄糖苷酶抑制剂中的用途。
4.根据权利要求1所述的肽在制备治疗糖尿病的药物中的应用。
5.根据权利要求1所述的肽在制备适用于糖尿病人群的降糖食品或保健品中的应用。
6.根据权利要求4或5所述的应用,其中所述糖尿病为2型糖尿病。
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