CN113005166B - 具有黄嘌呤氧化酶抑制活性的鳕鱼多肽 - Google Patents
具有黄嘌呤氧化酶抑制活性的鳕鱼多肽 Download PDFInfo
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- CN113005166B CN113005166B CN202110397871.1A CN202110397871A CN113005166B CN 113005166 B CN113005166 B CN 113005166B CN 202110397871 A CN202110397871 A CN 202110397871A CN 113005166 B CN113005166 B CN 113005166B
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- xanthine oxidase
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种具有黄嘌呤氧化酶抑制活性的鳕鱼多肽,其制备方法为:(1)取鳕鱼鱼排,烘干,粉碎;(2)加水制成底物液,加入蛋白酶,酶解得酶解液;所述蛋白酶选自木瓜蛋白酶、菠萝蛋白酶等;(3)酶解液通过G‑15葡聚糖凝胶层析柱分离洗脱,共出现5个洗脱峰,收集F2~F5中的任意一种或两种以上的洗脱液,冻干,即得。本发明还公开了具有黄嘌呤氧化酶抑制活性的小分子肽。本发明的鳕鱼多肽,具有黄嘌呤氧化酶抑制活性。本发明的小分子肽具有黄嘌呤氧化酶抑制(降尿酸)活性,其中YNVTGW还具有良好的DPPH和ABTS自由基清除活性,具有作为抑制尿酸水平和氧化应激的功能性产品的潜力。
Description
技术领域
本发明涉及一种具有黄嘌呤氧化酶抑制活性的鳕鱼多肽,属于酶解加工产品技术领域。
背景技术
鳕鱼鱼排是重要的鳕鱼加工副产品,含有人体必需氨基酸,且氨基酸的比例均衡,生物学效价较高,是优良的蛋白来源。然而其利用程度低,经济附加值低,且如果处理不当,将对环境造成严重威胁。
酶水解是从渔业副产物中有效回收蛋白质的方法之一,可用于改善和提升蛋白质的功能和营养特性,已被广泛应用于风味改善和生物活性肽的生产。
尿酸由黄嘌呤氧化酶依次催化次黄嘌呤和黄嘌呤氧化生成,尿酸的过量生产或排泄减少引发高尿酸血症。高尿酸血症是诸如慢性肾脏疾病、高血压、中风、动脉粥样硬化等健康问题的诱因。目前用于治疗高尿酸血症的药物往往伴随有严重的毒副作用,因此,开发生产成本低、毒副作用小、吸收程度高的食品源降尿酸肽意义重大。
发明内容
针对上述现有技术,为综合利用鳕鱼鱼排,本发明提供了一种具有黄嘌呤氧化酶抑制活性的鳕鱼多肽,其具有高附加值。本发明还提供了具有黄嘌呤氧化酶抑制活性的小分子肽。
本发明是通过以下技术方案实现的:
一种具有黄嘌呤氧化酶抑制活性的鳕鱼多肽,是通过以下方法制备得到的:
(1)取鳕鱼鱼排,烘干(65℃,20h),粉碎,得鳕鱼排粉,置于-20℃环境,备用;
(2)将鳕鱼排粉与水混合制成固液比为1:4~10(g:ml)的底物液,调节初始pH值为6~11(用氢氧化钠溶液或盐酸调节),按加酶量300~2400U/g(酶活/鱼排)加入蛋白酶,于45~60℃下震荡反应8h,得酶解液;所述蛋白酶选自木瓜蛋白酶、菠萝蛋白酶、中性蛋白酶、碱性蛋白酶或复合风味蛋白酶中的任意一种或两种以上。
进一步地,还包括步骤(3):所述酶解液通过G-15葡聚糖凝胶层析柱分离洗脱,流动相为超纯水,洗脱速度为2mL/min,共出现5个洗脱峰,收集F2~F5中的任意一种或两种以上的洗脱液,冻干,即得具有鳕鱼多肽产品。
优选的,所述步骤(2)中,固液比为1:5。
优选的,所述步骤(2)中,初始pH值为7.0。
优选的,所述步骤(2)中,加酶量为900U/g。
优选的,所述步骤(2)中,蛋白酶为碱性蛋白酶。
优选的,所述步骤(2)中,酶解温度为45℃。
优选的,所述步骤(2)中,酶解时控制振荡速率为200rpm。
优选的,所述步骤(3)中,G-15葡聚糖凝胶层析柱的规格为26mm×80cm。
优选的,所述步骤(3)中,收集F4或/和F5的洗脱液,经分离富集后的组分F4、F5,对XO的抑制率,较原酶解液、组分F2、组分F3,有显著提高。
小分子肽,其氨基酸序列为:Phe-Phe(FF)。
小分子肽,其氨基酸序列为:Tyr-Phe(YF)。
小分子肽,其氨基酸序列为:Trp-Pro-Trp(WPW)。
小分子肽,其氨基酸序列为:Trp-Pro-Asp-Ala-Arg-Gly(WPDARG),如SEQ ID NO.1所示。
一种小分子肽,其氨基酸序列为:Tyr-Asn-Val-Thr-Gly-Trp(YNVTGW),如SEQ IDNO.2所示。
经实验证明,上述5种小分子肽均具有黄嘌呤氧化酶抑制(降尿酸)活性,其中,SEQID NO.2所示的小分子肽不但具有黄嘌呤氧化酶抑制(降尿酸)活性,还具有良好的DPPH和ABTS自由基清除活性,该小分子肽具有作为抑制尿酸水平和氧化应激的功能性产品的潜力。
通过上述方法制得的鳕鱼多肽产品,具有黄嘌呤氧化酶抑制(降尿酸)活性。经LC-MS/MS质谱鉴定,组分F4中包括以下5条小分子肽:FF,YF,WPW,WPDARG和YNVTGW,这五条肽段均具有黄嘌呤氧化酶抑制(降尿酸)活性,其中YNVTGW还具有良好的DPPH和ABTS自由基清除活性。因此,本发明的鳕鱼多肽产品,以及小分子肽,具有作为抑制尿酸水平和氧化应激的功能性产品的潜力,可用于制备具有抑制尿酸水平功效的药物,可用于制备具有抑制氧化应激功效的药物,可用于制备具有抑制尿酸水平和氧化应激功效的药物。
本发明通过特定方法(酶解+凝胶过滤层析)处理鳕鱼鱼排,经LC-MS/MS鉴定,筛选得到了对黄嘌呤氧化酶具有高抑制活性的产物及肽段YNVTGW(IC50为0.23mM),该肽段YNVTGW还具有良好的DPPH和ABTS自由基清除活性,因此,该产品具有作为抑制尿酸水平和氧化应激的功能性产品的潜力。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1:蛋白酶对水解度、多肽转化率和黄嘌呤氧化酶抑制率的影响示意图,其中,A:水解度;B:多肽转化率;C:黄嘌呤氧化酶抑制率。
图2:加酶量对水解度、多肽转化率和黄嘌呤氧化酶抑制率的影响示意图,其中,A:水解度;B:多肽转化率;C:黄嘌呤氧化酶抑制率。
图3:固液比对水解度、多肽转化率和黄嘌呤氧化酶抑制率的影响示意图,其中,A:水解度;B:多肽转化率;C:黄嘌呤氧化酶抑制率。
图4:初始pH值对水解度、多肽转化率和黄嘌呤氧化酶抑制率的影响示意图,其中,A:水解度;B:多肽转化率;C:黄嘌呤氧化酶抑制率。
图5:酶解温度对水解度、多肽转化率和黄嘌呤氧化酶抑制率的影响示意图,其中,A:水解度;B:多肽转化率;C:黄嘌呤氧化酶抑制率。
图6:鳕鱼排酶解液G-15葡聚糖凝胶柱层析。
图7:样品质谱Basepeak图。
图8:FF、YF、WPW、WPDARG和YNVTGW的二级质谱图,其中,A、FF;B、YF;C、WPW;D、WPDARG;E、YNVTGW。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1鳕鱼鱼排酶解工艺优化
1.筛酶
选取木瓜蛋白酶、菠萝蛋白酶、中性蛋白酶、碱性蛋白酶和复合风味蛋白酶对原料进行酶解。准确称取预处理(鳕鱼鱼排在65℃下烘干20h,粉碎)后的鳕鱼鱼排5g,以固液比1:5配成底物液,按加酶量600U/g(酶活/鱼排)分别加入五种蛋白酶,在50℃,200rpm条件下反应8h后取样,灭酶,冷却,离心(8000rpm,10min),测定酶解液的XO抑制率、多肽含量和水解度(DH)。
游离氨基态氮的测定采用甲醛滴定法,总氮测定采用凯氏定氮法。取样品2mL样品,加入5mL蒸馏水,滴加1%酚酞溶液5滴,加入2mL甲醛,用0.05M NaOH溶液滴定至粉色,滴定所消耗的标定碱的物质的量即为体系中游离氨基的物质的量,从而测定发酵液的水解度。水解度计算公式如下:
A2=(V-V0)×C×14.008/2;
DH=A2/A1×100%;
式中:V和V0——样品组和空白组消耗的NaOH体积;A1——酶解液中总的氨基态氮;A2——不同水解时间,酶解液中游离氨基态氮含量;C——NaOH摩尔浓度。
多肽含量的测定:取200μL样品溶液,加入200μL 10%(W/V)的三氯乙酸(TCA)水溶液,于漩涡混合仪上混合均匀,静置10min,然后在4000rpm下离心10min,然后取300μL上述溶液置另一试管中,加入双缩脲试剂200μL(样液:双缩脲试剂=3:2,V/V),于漩涡混合仪上混合均匀,静置10min,4000rpm离心10min,取上清液于540nm下测定OD值,对照标准曲线求得样品溶液中的多肽浓度C(mg/mL),进而可求得样品中多肽含量。
体外XO抑制率的测定:分别将50uL待测样品,50uL 0.1U/mL的XO加入96酶标仪检测板中,37℃孵育10min后,加入150μL 0.4mmoL/L黄嘌呤溶液,酶促反应启动,同时记录2min内反应体系在290nm处吸光值的动力学变化,以pH 7.5PBS缓冲液做对照。根据公式计算样品对XO的抑制活性。
Ixo=(Vs-Vo)/Vo*100%;
式中:Ixo为样品的XO抑制率;Vo为酶促反应体系的初始反应速率;Vs为样品存在时酶促反应体系的初始速率。
本实验选择了五种蛋白酶对鳕鱼鱼排进行酶解,结果如图1所示。结果显示,碱性蛋白酶的酶解效果最佳,所得酶解液的水解度、多肽转化率和XO抑制率分别为13.81±0.12%、50.72±1.57%和50.49±5.33%,优于木瓜蛋白酶、菠萝蛋白酶、中性蛋白酶和复合风味蛋白酶。
2.加酶量优化
准确称取预处理后的鳕鱼鱼排5g,以固液比1:5配成底物液,按加酶量300、600、1200、1800、2400U/g(酶活/鱼排)加入碱性蛋白酶,在50℃,200rpm条件下反应8h后取样,灭酶,冷却,离心(8000rpm,10min),相关指标测定参照上述“筛酶”。
加酶量对水解度、多肽转化率及XO抑制率的影响如图2所示。随着加酶量的升高,酶解液的水解度呈现不断升高的趋势;而加酶量由600U/g增加至1200U/g时,酶解液多肽含量及XO抑制率并没有显著的提高(P>0.05),考虑到成本和效率,选择加酶量600U/g对工艺进一步优化。添加碱性蛋白酶600U/g时,酶解液DH、多肽转化率及XO抑制率分别为15.32±0.09%、57.20±1.06%和52.63±1.65%。
3.固液比优化
分别以固液比1:4、1:5、1:6、1:8、1:10配成底物液,按加酶量600U/g加入碱性蛋白酶,在50℃,200rpm条件下反应8h后取样,灭酶,冷却,离心(8000rpm,10min),相关指标测定参照上述“筛酶”。
固液比对水解度、多肽转化率及XO抑制率的影响如图3所示,其中以1:5组XO抑制率最高,为50.44±3.30%,显著高于1:4组(P<0.05)而与1:6组无显著性差异(P>0.05),此时,水解度和多肽转化率分别为14.57±1.47%和54.29±4.81%,故选择固液比1:5进行下面的优化。
4.初始pH值优化
准确称取预处理后的鳕鱼鱼排2.5g,以固液比1:5配成底物液,以1M HCl和1MNaOH调底物液的初始pH值为6、7、8、9、10、11,按加酶量600U/g加入碱性蛋白酶,在50℃,200rpm条件下反应8h后取样,灭酶,冷却,离心(8000rpm,10min),相关指标测定参照上述“筛酶”。
初始pH对水解度、多肽转化率、XO抑制率的影响如图4所示。初始pH为8时,酶解液的水解度和多肽转化率最高,分别为13.68±0.07和61.49±2.19%,此时酶解液的XO抑制率为45.09±3.07%,各指标与初始pH7无显著性差异(P>0.05),初始pH6时XO抑制率最高,为47.96±2.45%,与初始pH7、8无显著性差异(P>0.05)。而由于底物液的原始pH即为中性,故选择初始pH7进行下一步的优化。
5.酶解温度优化
准确称取预处理后的鳕鱼鱼排2.5g,以固液比1:5配成底物液,初始pH值为7,按加酶量600U/g加入碱性蛋白酶,分别在45、50、55、60℃,200rpm条件下反应8h后取样,灭酶,冷却,离心(8000rpm,10min),相关指标测定参照上述“筛酶”。
温度对水解度、多肽转化率、XO抑制率的影响如图5所示,水解度随温度的升高呈现先增长后降低的趋势,而多肽含量则随着温度升高缓慢升高。如图,随着酶解温度的升高,酶解液的XO抑制率呈微弱的下降趋势,但各组间无显著差异(P>0.05)。综合比较选择45℃作为最佳酶解温度。
实施例2凝胶过滤层析及其组分活性分析
葡聚糖凝胶G-15加入适量超纯水煮沸2h,除去浮沫及杂质;再加入超纯水搅拌、静置。除去上层杂质,重复以上操作直至上层无杂质。吸掉凝胶上层多余的水,将填料以玻璃棒引流至26mm×80cm层析柱,避免分层和气泡出现,柱子填好后用纯水冲洗,直至层析柱高度不再发生变化。
上样:以实施例1.5中制备的酶解液(酶解温度为45℃)作为样品进行上样。上样浓度及体积为:50mg/mL,8mL;收集出峰的组分(流动相为超纯水,洗脱速度为2mL/min),冻干测定不同组分的活性。
酶解液经凝胶色谱柱的分离结果如图6所示,依次收集洗脱峰F1~F5,冻干后复配成多肽浓度2mg/mL的溶液,测定各组分的XO抑制率,结果如表1所示。洗脱峰F4所对应的洗脱液的抑制率最高,因此选择洗脱峰F4所对应的洗脱液进行下一步的实验。
表1不同组分XO抑制率
实施例3F4肽段组成LC-MS/MS质谱鉴定
1.多肽提取:多肽组分脱盐后真空干燥,以0.1%三氟乙酸复溶,测定肽段浓度(OD280),以备LC-MS分析。
2.LC-MS/MS分析
样品取适量肽段使用纳升流速Easy nLC 1200色谱系统进行色谱分离。缓冲液:A液为0.1%甲酸水溶液,B液为0.1%甲酸、乙腈和水混合溶液(其中乙腈为80%)。色谱柱以100%的A液平衡。样品进样到Trap Column(100μm*20mm,5μm,C18,Dr.Maisch GmbH)后经过色谱分析柱(75μm*150mm,3μm,C18,Dr.Maisch GmbH)进行梯度分离,流速为300nL/min。
液相分离梯度如下:0min~3min,B液线性梯度从2%到8%;3min~43min,B液线性梯度从8%到28%;43min~51min,B液线性梯度从28%到40%;51min~52min,B液线性梯度从40%到100%;52min~60min,B液维持在100%。
肽段分离后用Q-Exactive HF-X质谱仪进行DDA(数据依赖采集)质谱分析。分析时长为60min,检测模式:正离子,母离子扫描范围:150~1800m/z,一级质谱分辨率:60 000@m/z 200,AGC target:3e6,一级Maximum IT:50ms。
肽段二级质谱分析按照下列方法采集:每次全扫描后触发采集20个最高强度母离子的二级质谱图谱(MS2 scan),二级质谱分辨率:15 000@m/z 200,AGC target:1e5,二级Maximum IT:25ms,MS2 Activation Type:HCD,Isolation window:1.6m/z,Normalizedcollision energy:28。
3.数据库检索
采用质谱数据库检索软件MaxQuant 1.6.1.0,蛋白质数据库Uniprot ProteinDatabase,物种Gadus(鳕属),共1833条蛋白序列,搜库分析参数设置如表2所示。
表2pFind分析参数设置
F4组分经质谱数据检索后采用PSM FDR≤0.01和Protein FDR≤0.01分别作为肽段与蛋白鉴定的筛选标准,得到724条肽段序列和27条蛋白序列,样品质谱Basepeak图如图7所示。
实施例4肽段筛选、合成及验证
1.配体的处理
采用Chem3D Pro 14.0软件绘制氨基酸、多肽的分子结构式,通过MM2力场优化,保存为mo12,并通过OpenBable 3.1.1软件转化为pdbq格式。
2.对受体处理
从PDB数据库中下载XO的晶体结构1N5X,采用PyMOL软件将1N5X中的B链删除,并抽离配体非布司他(TEI)保存为pdbqt格式备用。对受体1N5X去水加氢,计算gasteiger电荷,并将所有原子归属于AD4类型,保存为pdbqt格式备用。
3.分子对接
使用Autodock Vina(Vina)分子对接模拟氨基酸、多肽小分子与大分子蛋白质的相互作用。根据1N5X中原配体非布司他的位置,设置对接中心坐标为(96,54,39)(x,y,z),盒子大小为40×40×40,其它参数采取默认值。然后,将氨基酸和多肽配体逐一与XO晶体结构进行对接处理。
结果:本实施例基于黎青勇(黎青勇.核桃源降尿酸肽靶向抑制黄嘌呤氧化酶活性的构效机制研究:[博士学位论文].广州:华南理工大学,2018)及Nongonierma等(Nongonierma A B,Fitzgerald R J.Tryptophan-containing milk protein-deriveddipeptides inhibit xanthine oxidase.Peptides.2012,37(2):263~272)的研究,从实施例3中的724条肽段中选择了86条肽段与XO进行Autodock Vina模拟对接,根据Vina得分,我们筛选到Phe-Phe(FF)、Tyr-Phe(YF)、Trp-Pro-Trp(WPW)、Trp-Pro-Asp-Ala-Arg-Gly(WPDARG)和Tyr-Asn-Val-Thr-Gly-Trp(YNVTGW)五条肽段进行合成。
实施例5肽段合成及活性验证
1.肽段合成
肽段YF、WPW、WPDARG和YNVTGW均由生工生物工程(上海)股份有限公司采用Fmoc固相合成方法合成。
2.活性验证
XO抑制率
将多肽配成0.1~5mg/mL溶液,根据“筛酶”方法测定合成多肽的体外XO抑制率。
DPPH自由基清除率的测定
将合成肽段配成10mg/mL的多肽溶液,取0.5mL的样品,加入2.5mL 0.06mM的DPPH(溶于无水乙醇中),手动混匀,室温下避光反应0.5h,然后在517nm处测定其吸光值,对照组采用无水乙醇进行以上步骤,本实验以0.5g/L BHA为阳性对照。DPPH清除率计算如下:
DPPH清除率=(1-A样品/A空白)×100%;
式中:A样品——样品吸光值,A空白——为对照组吸光值。
ABTS自由基清除率的测定
将合成肽段配成10mg/mL的多肽溶液。ABTS自由基阳离子由含有ABTS储备溶液(7mM)和过硫酸钾(最终浓度为2.45mM)组成的混合溶液生成。将混合物在室温下于黑暗中孵育16小时,以使阳离子自由基显影,然后再使用去离子水稀释该自由基阳离子溶液,以在734nm处获得约0.70~0.72的吸光度。然后,吸取1.0mL将稀释的ABTS自由基溶液与1.0mL样品混合。30min后,在734nm处测量吸光度。每组实验重复三次。ABTS自由基清除活性的计算公式如下:
ABTS自由基清除率(%)=(Ac-As)/Ac×100%;
式中:As是样品的吸光度,Ac是对照的吸光度。
肽段的二级质谱图如图8所示,肽段的相关性质及活性验证结果如表3所示。
表3FF、YF、WPW、WPDARG和YNVTGW肽段信息与XO抑制率
注:(1)由于分子对接结果具有随机性,表中显示的Vina得分为10次对接结果(负值)最高值的平均值;
(2)YF、FF和WPW的疏水性(GRAVY)通过https://www.novopro.cn/tools/protein_gravy.html计算,WPDARG及YNVTGW的GRAVY值和稳定性通过https://web.expasy.org/protparam/计算;
(3)“-”表示未能计算稳定性/未检出活性。
如表3所示,五个肽段均具有较高的XO抑制活性,其中,YNVTGW表现出更高的XO抑制活性、DPPH自由基清除活性和ABTS自由基清除活性。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
序列表
<110> 中国海洋大学
<120> 具有黄嘌呤氧化酶抑制活性的鳕鱼多肽
<141> 2021-04-14
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 1
Trp Pro Asp Ala Arg Gly
1 5
<210> 2
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 2
Tyr Asn Val Thr Gly Trp
1 5
Claims (4)
1.一种小分子肽,其氨基酸序列为:Trp-Pro-Asp-Ala-Arg-Gly,如SEQ ID NO.1所示。
2.权利要求1所述的小分子肽在制备具有抑制尿酸水平功效的药物中的应用;或:在制备具有抑制氧化应激功效的药物中的应用;或:在制备具有抑制尿酸水平和氧化应激功效的药物中的应用。
3.一种小分子肽,其氨基酸序列为:Tyr-Asn-Val-Thr-Gly-Trp,如SEQ ID NO.2所示。
4.权利要求3所述的小分子肽在制备具有抑制尿酸水平功效的药物中的应用;或:在制备具有抑制氧化应激功效的药物中的应用;或:在制备具有抑制尿酸水平和氧化应激功效的药物中的应用。
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