CN114716524B - 抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽 - Google Patents
抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽 Download PDFInfo
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- CN114716524B CN114716524B CN202210396241.7A CN202210396241A CN114716524B CN 114716524 B CN114716524 B CN 114716524B CN 202210396241 A CN202210396241 A CN 202210396241A CN 114716524 B CN114716524 B CN 114716524B
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- glucosidase
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Abstract
本申请提供了抑制α‑淀粉酶和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.1‑6。本申请的肽能够同时有效抑制α‑葡萄糖苷酶和α‑淀粉酶活性，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，本申请提供了多个抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽及其应用和筛选方法。
背景技术
糖尿病已经成为一种全球流行的代谢疾病，主要表现为血糖水平升高引起的机体脂肪、碳水化合物和蛋白质代谢紊乱。近年来，糖尿病患病率一直在稳步增长。非胰岛素依赖型糖尿病即2型糖尿病常见发生于各个年龄段的人群中，目前90％的糖尿病患者已被诊断为2型糖尿病。膳食淀粉可以被α-淀粉酶消化成大量的麦芽糖，然后再被α-葡萄糖苷酶消化为葡萄糖。淀粉快速降解为葡萄糖导致的血糖水平升高被称为餐后高血糖症，这是2型糖尿病的重要指标。因此可以通过抑制α-淀粉酶和α-葡萄糖苷酶，延迟碳水化合物水解为葡萄糖的速率来实现控制餐后血糖的目的。目前用于治疗2型糖尿病的市售药物，例如二甲双胍，阿卡波糖、伏格列波糖和米格列酮可以抑制α-淀粉酶和α-葡萄糖苷酶活性，但摄入这些降血糖药物也会带来一些副作用，例如胀气和腹泻等。
近年来，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。食物蛋白质作为功能肽的主要来源受到了广泛关注。小米醇溶蛋白已被证实具有较好的改善血糖代谢的功能，也就是说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。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，但尚未有关于小米蛋白降糖肽的相关研究。因此，有必要探究小米醇溶蛋白水解得到的小分子肽中可以作为潜在的功能性降糖物质的肽段。
发明内容
针对以上问题，一方面，本申请提供抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽，所述肽的序列选自SEQ ID NO.1-6。
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，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-淀粉酶和/或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，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-葡萄糖苷酶和α-淀粉酶对接效果较好的肽段；比对分别与α-葡萄糖苷酶和α-淀粉酶对接效果较好的肽段，得到同时能与α-葡萄糖苷酶和α-淀粉酶有较好对接效果的肽段；
(3)活性位点分析及体外功能预测：使用殷赋云平台对筛选得到的肽序列分别与α-淀粉酶和α-葡萄糖苷酶进行对接，确定其与α-淀粉酶和α-葡萄糖苷酶作用的关键氨基酸及相互作用力；然后对筛选得到的同时能与α-葡萄糖苷酶和α-淀粉酶有较好对接效果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。
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：
本次首次从熟小米醇溶蛋白中筛选得到了能够同时有效抑制α-葡萄糖苷酶和α-淀粉酶活性的6条未经报道的小肽QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK，并明确了小肽QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK的结构；同时QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK具有安全无毒无副作用的优点，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QQLRPF与α-葡萄糖苷酶和α-淀粉酶对接结果图。
图2为AGAGPQGRP与α-葡萄糖苷酶和α-淀粉酶对接结果图；
图3为FALQGAAFLGSA与α-葡萄糖苷酶和α-淀粉酶对接结果图；
图4为QQQQLLR与α-葡萄糖苷酶和α-淀粉酶对接结果图；
图5为KTGSGAEGMHGGK与α-葡萄糖苷酶和α-淀粉酶对接结果图；
图6为KAHAALGAK与α-葡萄糖苷酶和α-淀粉酶对接结果图；
图7为QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK的α-葡萄糖苷酶抑制率图；
图8为QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK的α-淀粉酶抑制率图。
具体实施方式
实施例1熟小米醇溶蛋白的提取
首先将小米磨成粉，并通过60目筛。然后将小米粉以1:5(w/v)的比例分散在正己烷中,37℃水浴振荡4h后静置，待小米粉和正己烷明显分层后，倒去上层正己烷并收集下层沉淀物，在通风橱中干燥24h以完全除去正己烷，所得的小米粉即为生小米粉。将脱脂生小米粉与蒸馏水按1:5的比例混合均匀,在沸水浴中糊化10min后，将沉淀物在烘箱43℃下干燥24h，然后打粉并通过60目筛网过筛即可得到熟小米粉。将熟小米粉以1:7(m/v)的比例分散在70％乙醇溶液中，然后设置温度为40℃，在水浴振荡器中振荡2h。之后将上述反应溶液在7000rpm下离心10min，并收集上清液，用透析袋透析24h。透析过程中更换蒸馏水4次。透析结束后将透析液在7000rpm下离心5min，收集沉淀，冻干后得到熟小米醇溶蛋白，蛋白含量为85％。
实施例2熟小米醇溶蛋白的体外模拟消化
将熟小米醇溶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熟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熟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-115kcal/mol和内部排斥能小于30kcal/mol对测序得到的肽段分别与α-葡萄糖苷酶和α-淀粉酶进行虚拟筛选，筛选结果如表1和表2所示。对比表1和表2可以找出，小肽QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK同时跟α-葡萄糖苷酶和α-淀粉酶具有较好的对接效果。
表1虚拟筛选中与α-淀粉酶对接效果较好的肽段
表2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蛋白/核酸/多肽小分子对接Dock 6.9软件进一步进行精确分子对接分析，找出QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK与α-葡萄糖苷酶和α-淀粉酶作用的活性位点。如图1A-B所示，分析QQLRPF与α-淀粉酶的相互作用力发现，QQLRPF与α-淀粉酶的残基Trp 59、Leu 165、Trp 58、Try 62、Ile235和His 305形成了疏水相互作用。除此之外，残基His 305以及Gln 63与QQLRPF之间形成了氢键。肽段QQLRPF还分别与α-淀粉酶的残基Glu 233和His 101之间形成了盐桥，也就是离子键。分析QQLRPF与α-葡萄糖苷酶的相互作用力发现，QQLRPF与α-葡萄糖苷酶的残基Trp481、Ala 555、Trp 516、Trp 376、Leu 677、Leu 678、Leu 650、Phe 297、Val 334、Ala 343和Thr 339形成疏水相互作用，与Thr 339、Arg 429、Glu 427、Gly 399、Asn 301、Gly 651和Ser 676形成氢键，与Asp 282、Asp 616和Glu 377形成盐桥，与Phe 649形成π-π堆积(图1C-D)。如图2A-B所示，AGAGPQGRP与α-淀粉酶的残基Arg 195、His 299和Asp 356形成盐桥，与残基Leu 237、Glu 233和His 201形成氢键，与Tyr 62、Trp 58、Ile 235和Leu 165形成疏水相互作用。此外，AGAGPQGRP与α-葡萄糖苷酶的残基His 105形成盐桥，与Tyr 65和Phe 166形成疏水相互作用，与Lys 225和Gly 228形成氢键(图2C-D)。如图3A-B所示，FALQGAAFLGSA与α-淀粉酶的残基Tyr 151、Leu 165、Tyr 62、Trp 58、Trp 59和Ile 51形成疏水相互作用，与Lys 200形成盐桥，与His 305、Asp 300、Thr 163、Tyr 52和Gln 63形成氢键,与Trp 357形成π-阳离子相互作用，与Tyr 62形成π-π堆积。分析FALQGAAFLGSA与α-葡萄糖苷酶的相互作用力可知，FALQGAAFLGSA与α-葡萄糖苷酶的残基Thr 203、Phe 166、Phe 147、Lys 398、Val 335和Glu 377形成疏水相互作用，与Glu 271和His 304形成盐桥，与Gly 228和Ala348形成氢键(图3C-D)。如图4A-B所示，QQQQLLR与α-淀粉酶的残基Asp 353、Asp 356、Gly304、His 299和Glu 233形成氢键，与His 305、Asp 197和Glu 233形成盐桥，与His 305、Ala198、Trp 58和Tyr 62形成疏水相互作用,与Tyr 62形成π-阳离子相互作用。此外，QQQQLLR与α-葡萄糖苷酶的残基Asp 62、Gly 228、His 332和Gly 399形成氢键，与Asp 202、Asp 333和Glu 271形成盐桥，与Phe 166、Tyr 389、Arg 400、Val 334、Phe 297、Phe 397和Glu 231形成疏水相互作用(图4C-D)。接下来首先对α-淀粉酶与肽段KTGSGAEGMHGGK结合作用力进行分析，如图5A-B所示。肽段KTGSGAEGMHGGK与α-淀粉酶残基Trp 284和Tyr62形成了疏水作用。此外，α-淀粉酶残基Lys 200、Lys 225、Lys 261、Gly 283均与KTGSGAEGMHGGK之间以氢键相连接。除疏水相互作用和氢键外，残基His 201与肽段KTGSGAEGMHGGK之间形成了π-π堆积，而残基Trp284则以π-阳离子相互作用与肽段KTGSGAEGMHGGK连接。此外，His 101、Glu233和Glu 272均与肽段KTGSGAEGMHGGK形成了盐桥，也就是离子键。如图5C-D所示，KTGSGAEGMHGGK与α-葡萄糖苷酶的残基Trp 237和Lys 221形成了疏水相互作用。此外，α-葡萄糖苷酶的残基Gly 399、Lys 225和Ala 343均与KTGSGAEGMHGGK之间以氢键相连接。除疏水相互作用和氢键外，肽段KTGSGAEGMHGGK还与α-葡萄糖苷酶的残基Arg 429形成盐桥，也就是离子键。最后对肽段KAHAALGAK与α-淀粉酶结合后的作用力进行分析，如图6A-B所示。肽段KAHAALGAK与α-淀粉酶残基Leu 235、Ala 198、Leu 165、Tyr 62、Ile 51、Trp 59之间形成了疏水作用，与残基Gln 63、Thr 163之间形成了2个氢键，与残基Glu 240形成了盐桥(离子键)，与残基His 201以π-π堆积作用力相连接。然后对肽段KAHAALGAK与α-葡萄糖苷酶结合后的作用力进行分析，如图6C-D所示，KAHAALGAK与α-葡萄糖苷酶残基Glu 377、Thr 339、Val 334、Val 335、Phe 397和Ala 224之间形成疏水相互作用，与残基Arg 429、Asn 301、Lys 225和Gly 222之间形成了4个氢键，与残基Asp 379形成了盐桥。QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK的稳定性通过EXPASY平台(http://web.expasy.org/protparam/)评估。利用admetSAR预测QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK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3所示。可以看到，六条小肽均具有低毒性，且KTGSGAEGMHGGK、KAHAALGAK和FALQGAAFLGSA三条肽段具有良好的体外稳定性，而QQLRPF、KTGSGAEGMHGGK和KAHAALGAK则具有良好的人体肠道吸收特性。此外，除FALQGAAFLGSA具有疏水特性且呈酸性外，QQLRPF、AGAGPQGRP、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK均具有较好的亲水性且呈碱性，并且AGAGPQGRP的半衰期也较长。
表3肽段QQLRPF、AGAGPQGRP、FALQGAAFLGSA、QQQQLLR、KTGSGAEGMHGGK和KAHAALGAK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-淀粉酶和α-葡萄糖苷酶抑制活性测定
α-淀粉酶活性测定方法如下所示：500μL样品(0.5％)和500μL 0.02mol/L磷酸钠缓冲液(pH＝6.9，0.006mol/L NaCl，包括α-淀粉酶溶液(13U/mL))在25℃下孵育10min，预孵育后加入500μL 1％可溶性淀粉溶液(0.02mol/L磷酸钠缓冲液，pH＝6.9，0.006mol/LNaCl)，在25℃下孵育10min，并加入1.0mL DNS试剂，之后将上述溶液在沸水浴中煮沸5min，停止反应并冷却到室温。反应液用10mL蒸馏水稀释并在540nm处读数。对照选用去离子水替换样品。计算公式如下:
α-葡萄糖苷酶活性测定方法如下所示：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7所示，可以看到QQLRPF和AGAGPQGRP的α-葡萄糖苷酶抑制率显著高于KTGSGAEGMHGGK、KAHAALGAK、FALQGAAFLGSA和QQQQLLR，分别为32.67％和31.57％。FALQGAAFLGSA的α-葡萄糖苷酶抑制率为17.67％，显著高于KTGSGAEGMHGGK(15.64％)、KAHAALGAK(13.77％)和QQQQLLR(15.94％)，KAHAALGAK抑制率最低。如图8所示，可以看到QQLRPF的α-淀粉酶抑制率最高，为42.67％，其次为AGAGPQGRP，即37.67％，然后是FALQGAAFLGSA，为24.42％。最后是KTGSGAEGMHGGK、KAHAALGAK和QQQQLLR，分别为15.66％、15.42％和16.99％。
SEQUENCE LISTING
<110> 中国农业大学
<120> 抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽
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Claims (7)
1.抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽，其特征在于，所述肽的序列选自SEQ ID NO.1-6。
2. 根据权利要求1所述的肽，其中所述肽的序列为SEQ ID NO.1或SEQ ID NO.2。
3.组合物，其特征在于，所述组合物包含根据权利要求1或2所述的肽，以及药学、食品或保健品上可接受的辅料。
4.根据权利要求1或2所述的肽在制备α-淀粉酶和/或α-葡萄糖苷酶抑制剂中的用途。
5.根据权利要求1或2所述的肽在制备治疗糖尿病的药物中的应用。
6.根据权利要求1或2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。
7.根据权利要求5或6所述的应用，其中所述糖尿病为2型糖尿病。
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