摘要
食源性二肽基肽酶Ⅳ(Dipeptidyl peptidase-Ⅳ,DPP-Ⅳ)抑制肽和抗氧化肽分别有助于清除体内自由基和调节机体血糖。为实现脱脂南极磷虾粉的高值化利用,以及为糖尿病治疗寻找新方法,本研究从脱脂南极磷虾粉酶解产物中制备DPP-Ⅳ抑制肽和抗氧化肽段。使用碱性蛋白酶、中性蛋白酶和胰酶水解脱脂南极磷虾粉,探究时间与水解度、活性、产率的关系,并分离纯化多肽,最后通过分子对接确定抑制作用位点。结果表明,酶解液经超滤、Sephadex G15分离纯化和液相-质谱联用技术鉴定后,得到了4条具有DPP-Ⅳ抑制和抗氧化作用的肽,氨基酸序列分别为WPPLSPFRCPR、IPDWFLNRQ、FLWLKKTPLPL和DTVPWFPR。其中IPDWFLNRQ具备较高的DPP-Ⅳ抑制活性,DPP-Ⅳ抑制活性IC50值为(0.616±0.097) mmol/L;WPPLSPFRCPR具有较高的抗氧化活性,DPPH EC50值为(0.098 3±0.011) mmol/L,ABTS EC50值为(0.116±0.073) mmol/L。分子对接结果表明这4个肽主要通过氢键与DPP-Ⅳ活性中心及其以外的位点相结合,从而达到抑制DPPP-Ⅳ活性的作用。本研究基于脱脂南极磷虾粉制备食源性DPP-Ⅳ抑制肽和抗氧化肽,以期为南极磷虾粉的开发利用提供理论依据。
关键词
糖尿病是一种以高血糖为主要特征的代谢性疾病,会引发肾功能衰竭、心脏病变等糖尿病并发症,多数是由于患者体内活性氧自由基水平升高,导致氧化应激而引发
南极磷虾(Euphausia superba)生物资源量为3.42亿~3.56亿t。南极磷虾氨基酸种类丰富、蛋白质含量高,是世界上最大的动物蛋白质资源,其蛋白质中含有8种必需氨基酸,被认为是未来海洋食品和保健品中最合适和最丰富的资
因此,本研究以脱脂南极磷虾粉为原料,通过酶解法提取多肽,以ABTS和DPPH自由基清除能力以及DPP-Ⅳ抑制能力为标准,采用超滤和凝胶层析技术进行纯化。最后对活性肽进行结构鉴定,并通过分子对接研究DPP-Ⅳ抑制肽的抑制机理,以期为脱脂南极磷粉的高值化利用和降血糖、抗氧化药物的开发提供理论依据。
脱脂南极磷虾粉由山东康境海洋生物工程有限公司(山东,中国)于2022年2月提供。其中,水分含量为5.98%,粗蛋白含量为72.26%,粗脂肪含量为3.02%,灰分含量为10.23%。碱性蛋白酶、胰蛋白酶、中性蛋白酶均来自天津诺奥酶促有限公司(天津,中国)。DPP-Ⅳ抑制剂筛选试剂盒、邻苯二甲醛(OPA)、十二烷基硫酸钠(SDS)、二硫苏糖醇(DTT)均购自西格玛奥德里奇(上海)贸易有限公司(上海,中国)。其他试剂均为分析级,来自北京百灵威科技有限公司(北京,中国)。
主要仪器与设备:GL-21M 高速冷冻离心机,上海卢湘仪离心机仪器有限公司;HDB-7L核酸蛋白检测仪、CBA-A程控多功能全自动部分收集器,上海沪西分析仪器有限公司;FD-1PF冷冻干燥机,北京德天游有限公司;FLEXSTATION3多功能读板机,美国Molecular Devices公司;ULTIMATE3000毛细管高效液相色谱仪、Q EXACTIVE四级杆-静电场轨道阱高分辨液相色谱-质谱联用仪,赛默飞世尔科技有限公司。
脱脂南极磷虾粉放入8倍体积的蒸馏水中,水浴加热至55 ℃保持30 min,然后加入碱性蛋白酶、胰酶和中性蛋白酶,每种酶的剂量为1%(质量分数)。在分别反应1、2、3、4、5、6 h后,将水解物取出再次加热到90 ℃保持20 min,使酶失活,然后离心30 min(10 000g,4℃)。上清液冻干后在-20 ℃保存(最长保存期限为3个月)。在冻干后,肽的得率(Y)的计算公式:
(1) |
式中:W1和W2分别为冻干后肽粉的质量和虾粉蛋白的质量,g。
参考TANG
(2) |
式中:h为通过甘氨酸标准曲线计算断裂肽键数;htot为单位重量的肽键总数。
将肽溶解在Tris-HCl缓冲液(0.1 mol/L, pH 8.0)中制备样品溶液,50 μL DPP-Ⅳ (10 U/L)和25 μL样品在96孔板37 ℃孵育10 min。然后加入25 μL Gly-Pro-pNA(1.6 mmol/L)底物,37 ℃孵育15 min后测定其荧光强度。DPP-Ⅳ抑制率(WDPP-Ⅳ)计算公式:
(3) |
式中:Fcontrol为对照组的荧光值;Fsample为样品组的荧光值;Fblank为空白组的荧光值。
首先,将1.5 mL肽溶液(5 mg/mL)与等体积5 mg/100 mL浓度的DPPH溶液混合,并在避光的环境中保存30 min。使用紫外分光光度计在517 nm测量。DPPH自由基清除率(WDPPH)计算公式:
% | (4) |
式中:Asample为样品组的吸光度;Ablank为空白组的吸光度。
7 mmol/L ABTS与2.45 mmol/L过硫酸钾混合,在黑暗中保存16 h后,用0.2 mol/L PBS (pH 7.4)稀释该混合物制备ABTS溶液,此时ABTS溶液的吸光度在734 nm处为0.70 ± 0.02。然后,将30 μL肽溶液(5 mg/mL)与3 mL ABTS溶液混匀,在避光的环境中放置6 min。然后在734 nm处读取吸光度。ABTS自由基清除活性(WABTS)计算公式:
(5) |
式中:Asample为样品组的吸光度;Ablank为空白组的吸光度。
脱脂南极磷虾粉酶解液经过超滤,在不同的分子量≥10 ku, 10 ku>MW≥5 ku, 5 ku>MW≥3 ku和<3 ku范围内收集,收集的4个组分被浓缩、冻干并保存在-20 ℃进一步分析。
将目标超滤组分溶于去离子水(20 mg/mL)中。将5 mL样品注入Sephadex G-15凝胶柱然后使用去离子水洗脱样品。按照每6 min收集一次,用核酸蛋白检测仪在254 nm下测定样品洗脱曲线的吸光度。肽段按照得到的6个峰进行分组。收集不同的组分,冻干,然后存储在-20 ℃以进一步分析。
通过同源对比和检索从PDB数据库(http://www.rcsb.org/pdb)获取DPP-Ⅳ(PDB ID:5y7h)的三维晶体结构,使用AutoDock软件对DPP-Ⅳ受体蛋白进行处理,多肽的3D结构采用PyMol 2.5.4构建,将多肽配体和DPP-Ⅳ受体进行对
通过PeptideRanker软件(http://distilldeep.ucd.ie/PeptideRanker)预测多肽的潜在生物活性,使用iDPPIV-SCM软件(http://camt.pythonanywhere.com/iDPPIV-SCM)对多肽的DPP-Ⅳ抑制活性进行评分,根据CONWAY
水解度能反映肽键的裂解程度,与肽链的长度有关,也会影响其结构中氨基酸的暴露情况和生物活性。如
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图1 酶解时间与水解度之间的关系
Fig.1 Effect of hydrolysis time on degree of hydrolysis
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference (P > 0.05), while with different lowercase letters mean significant difference(P < 0.05).
脱脂南极磷虾粉酶解液的活性测定见
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图2 酶解时间与肽得率之间的关系
Fig.2 Effect of hydrolysis time on peptides yield
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).

图3 脱脂南极磷虾水解物酶解时间与DPPH和ABTS自由基清除活性的关系(5 mg/mL)
Fig.3 Effect of hydrolysis time on DPPH and ABTS free radical scavenging of Antarctic krill hydrolysates (5 mg/mL)
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).

图4 酶解时间与肽得率之间的关系脱脂南极磷虾水解物酶解时间与DPP-Ⅳ抑制活性的关系(20 mg/mL)
Fig.4 Effect of hydrolysis time on DPP-Ⅳ inhibitory activity of Antarctic krill hydrolysates (20 mg/mL)
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).
超滤是一种根据溶液中分子大小来分离溶液中分子的技
与超滤不同的是葡聚糖凝胶过滤可以以更精细的方式分离不同分子量的水解物。在本实验中,分子量<3 ku的馏分通过Sephadex G-15柱后被分离成6个组分(M1、M2、M3、M4、M5和M6,见
超滤组分 Ultrafiltration fractions | DPP-Ⅳ抑制率20 mg/mL DPP-Ⅳ inhibitory activity /% | DPPH自由基清除活性5 mg/mL DPPH free radical scavenging /% | ABTS自由基清除活性5 mg/mL ABTS free radical scavenging /% |
---|---|---|---|
≥10 ku |
45.67±1.2 |
16.73±2.1 |
27.48±1.1 |
10 ku>MW≥5 ku |
52.73±0.1 |
34.68±0.7 |
20.37±0.7 |
5 ku>MW≥3 ku |
70.94±0.7 |
42.29±1.2 |
40.33±2.2 |
< 3 ku |
83.12±0.7 |
55.40±0.4 |
40.25±1.1 |
注: 相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Notes: Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).

图5 Sephadex G-15凝胶色谱图
Fig.5 Sephadex G-15 gel chromatogram
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图6 各组分DPP-Ⅳ抑制活性
Fig.6 Inhibition activity of DPP-Ⅳ of fraction
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).
通过LC-MS/MS共鉴定了128条来自M3组分的肽。在所有的肽段中,根据肽段的MS/MS谱的得分、PeptideRanker软件的预测分数、iDPPIV-SCM软件评分并根据CONWAY
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图7 各组分DPPH自由基清除活性
Fig.7 DPPH radical scavenging activity of fractions
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).

图8 各组分ABTS自由基清除活性
Fig.8 ABTS radical scavenging activity of fractions
相同小写字母表示差异不显著(P > 0.05),不同小写字母表示差异显著(P < 0.05)。
Values marked with the same lowercase letter mean no significant difference(P > 0.05), while with different lowercase letters mean significant difference (P < 0.05).
多肽的生物活性往往与它们所含的特定氨基酸的比例和这些氨基酸的位置有
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图 9 M3组分的6条多肽MS/MS图谱
Fig.9 Sequence profile of six of the peptides identified from M3 fraction
一般来说,具有高疏水性氨基酸残基的肽可以抑制DPP-Ⅳ的活
在这项研究中,AutoDock Vina软件用来模拟DPP-Ⅳ单体与4个选定的肽的对接。亲和力分数是基于各种因素,包括空间效应、排斥、氢键、疏水相互作用和受体-配体复合物之间的灵活性,反映了配体与受体有效结合的可能性。较低的对接分数表明肽和DPP-Ⅳ之间有更好的结合构
肽序列 Peptide sequence | 分子量 Molecular weight/u | DPP-IV IC50 /(mmol/L) | DPPH EC50 /(mmol/L) | ABTS EC50/(mmol/L) |
---|---|---|---|---|
WPPLSPFRCPR | 1 355 | 2.121±0.31 | 0.098 3±0.011 | 0.116±0.073 |
NRPIPPWI | 991.6 | >5 | 1.249±0.044 | 4.123±0.067 |
IPDWFLNRQ | 1 188 | 0.616±0.97 | 3.083±0.038 | 1.466±0.040 |
PFGLYVHHSWF | 1 389 | >5 | 1.416±0.076 | >5 |
FLWLKKTPLPL | 1 355 | 1.158±0.71 | 2.083±0.053 | 4.663±0.022 |
DTVPWFPR | 1 017 | 4.923±2.0 | 0.802±0.078 | 2.471±0.043 |
蛋白 Protein | 结合能 Binding energy/(kcal/mol) | |||
---|---|---|---|---|
DPP-Ⅳ | WPPLSPFRCPR | IPDWFLNRQ | FLWLKKTPLPL | DTVPWFPR |
-8.4 | -8.6 | -8.4 | -8.3 |
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图10 四条肽与DPP-Ⅳ结合的3D图
Fig.10 3D diagrams of the combination of the four superior peptides and DPP-Ⅳ
该研究首次从脱脂南极磷虾粉中提取并鉴定出具有双重生物活性的肽序列。研究得出,低分子量的肽组分具有更强的DPP-Ⅳ抑制活性和抗氧化活性。通过超滤、Sephadex G-15和LC-MSMS,从活性最高的组分中确定了4种新的、高效的生物活性肽。分子对接证实4种肽都能通过氢键与DPP-Ⅳ的活性部位结合。因此,从脱脂南极磷粉中提取的多肽可以为降血糖、抗氧化药物的开发提供理论依据。
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