CN113667653B - 基于超氧化物歧化酶的透皮全吸收型抗衰老纳米功能面膜 - Google Patents
基于超氧化物歧化酶的透皮全吸收型抗衰老纳米功能面膜 Download PDFInfo
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Abstract
本发明提供了一种新的超氧化物歧化酶突变体，该突变体显示出改善的康皮肤衰老、减少皱纹的用途，具有广阔的应用前景。进一步地，本发明还提供了包含该突变体的透皮全吸收型抗衰老纳米功能面膜。
Description
技术领域
本发明属于生物酶领域，更为具体的，本发明涉及突变的超氧化物歧化酶及基于该超氧化物歧化酶的透皮全吸收型抗衰老纳米功能面膜。
背景技术
导致皮肤老化的两个主要原因是由于太阳光照射和活性氧的侵入。活性氧在去除进入体内的异物的过程中通过分解异物来去除异物。然而在起到积极的作用的同时，如果由于各种原因产生过多的活性氧，不仅侵害自身的细胞组织，而且与体内的不饱和脂肪酸结合而形成过氧化脂质。尤其，在皮肤中与蛋白质、脂质结合而生成氧化物，从而导致皮肤老化，而且对黑色素的氧化过程起到较强的作用，引发色素沉着。
超氧化物歧化酶(Superoxide Dismutase，SOD)是生物体内存在的一种抗氧化金属酶，它能够催化超氧阴离子自由基歧化生成氧和过氧化氢，在机体氧化与抗氧化平衡中起到至关重要的作用。根据SOD所含金属辅基的不同，一般可将其分为Cu，Zn-SOD、Mn-SOD、Fe-SOD三种类型。外源SOD的补充具有延缓皮肤衰老、抗氧化、祛色斑的功用，广泛添加于各类化妆品或护肤品中。
我国自上世纪八十年代以来，对SOD的应用研究主要侧重于动植物SOD的提取和纯化，国内SOD产品大部分是从动物血液中制备的，但由于原材料有限及卫生安全性等原因，导致制品产量有限，特别是随着世界各地疯牛病、禽流感、***等恶性传染病的不时报道，动物源血液制品的风险加大，纯度要求的增高也增加了产品的成本，特别是由于疯牛病的影响，欧盟规定从1997年1月起，禁止使用牛血SOD作为食品添加剂使用后，利用基因工程是广开SOD酶源、降低成本和获得具有天然活性的SOD的有效途径。但SOD酶存在基因工程产量低、活性不稳定等问题，并且应用于化妆品时需要增加其对温度更为广泛的稳定性从而便于运输，因此开发一种能够克服上述不利因素的SOD酶就显得十分必要。
发明内容
为了解决上述技术问题，本申请的第一方面提供了一种突变的Cu，Zn-SOD酶。更为具体的，所述突变的Cu，Zn-SOD酶的氨基酸序列如SEQ ID NO.1所示。
本申请的第二方面，提供一种突变的Cu，Zn-SOD酶在用于制备抗衰老化妆品中的应用，优选的，所述化妆品为精华液或面膜，更为优选的，所述精华液包括以下质量百分比的组分：
所述的突变的Cu，Zn-SOD酶2-5％、甘油5-8％、透明质酸0.01-2％、左旋维生素C0.01-2％、维生素E 2-5％、尿素1-2％，丙二醇0.5-2％，余量为去离子水。
本申请的第三方面，提供一种含有所述突变的Cu，Zn-SOD酶的抗衰老面膜，所述面膜的制备方法为利用所述精华液浸泡面膜材料或添加于面膜材料中。更为优选的，所述面膜为纳米面膜。
与现有技术相比，本发明具有以下优点及有益效果：
1)本发明通过对酿酒酵母(Saccharomyces cerevisiae)Cu，Zn-SOD酶(NP_012638.1)进行氨基酸突变，得到了H47A,G86W双突变Cu，Zn-SOD酶，在活力保持的情况下，热稳定性有了明显提高，具有广泛的应用价值和经济价值；
2)本发明通过基因工程技术发酵生产突变的Cu，Zn-SOD酶，并将其制备为精华液及面膜等护肤制品，所述制品能使肌肤恢复有弹性的年轻态，具有显著的抗衰效果。
附图说明
图1各突变体在热处理前后的酶活对比
具体实施方式
实施例1：突变体酶热稳定性
由NCBI中获得来自于酿酒酵母(Saccharomyces cerevisiae)Cu，Zn-SOD酶(NP_012638.1)，选择其活性位点上的4个关键氨基酸位点(S39G、H47A,G86W，H131Q)进行突变改造研究。
利用一步反向PCR方法构建Cu，Zn-SOD酶定点改造突变体，测序验证后，将正确构建的定点突变改造质粒转化到宿主细胞中。
将正确构建的各突变位点(组1：S39G+H47A、组2：S39G+G86W、组3：S39G+H131Q、组4：H47A+G86W、组5：H47A+H131Q、组6：G86W+H131Q)重组菌经平板划线分离出单菌落，挑取单菌落至LB培养基(Kan)中培养12h，接种至TB发酵培养基(Kan)中发酵培养26h，取发酵上清即各突变重组酶在pH7.0,60℃水浴保温热处理15min，分别检测热处理前后酶活。以未突变且未在60℃热处理的原始酶酶活作为100％(control组)。结果如图1所示，S39G+H47A、H47A+G86W突变体经热处理后，剩余酶活高于对照菌。H47A+G86W突变体表现出显著的热稳定性的提升。
实施例2：抗氧化活性验证
测试样品：野生型Cu，Zn-SOD酶和H47A+G86W双突变Cu，Zn-SOD酶分别用超纯水混合从稀释成质量分数为5％的测试溶液。
羟自由基清除实验：利用本领域常规的分光光度法，在536nm下分别测量各组混合溶液的吸光度值，记为As，以蒸馏水代替样品溶液作为空白组测吸光度值，记为Ab，以蒸馏水替代H2O2作为损伤组，测其吸光度值An，测试样品羟自由基清除率按以下公式计算：羟自由基清除率(％)＝[(As-An)/(Ab-An)]×100％，取三次测量的平均值。测试结果如表1所示。
DPPH自由基清除实验：取3mL浓度为0.2mmol/L的DPPH-无水乙醇溶液于比色皿中，再加入1mL测试样品，混匀，室温避光30min，在517nm波长处测吸光度，吸光值记为A1，加入3mL无水乙醇溶液和1mL测试样品溶液的吸光度值记为A2，加3mL DPPH-无水乙醇溶液和1mL无水乙醇溶液的吸光值记为A3。按下式计算DPPH自由基清除率：DPPH自由基清除率(％)＝[1-(A1-A2)/A3]×100％，取三次测量的平均值。测试结果如表1所示。
表1
从表1的实验结果可以看出，本发明H47A+G86W双突变Cu，Zn-SOD酶在羟自由基清除率和DPPH自由基清除率上相对于野生型均有提升，说明该改善热稳定性的突变体Cu，Zn-SOD酶可有效清除自由基，具有抗氧化、抗衰老的功效。
实施例3突变体制品皮肤弹性和保湿功能测试
将上述H47A+G86W双突变Cu，Zn-SOD酶制备为护肤精华液，所述护肤精华液包括以下质量百分比的组分：
H47A+G86W双突变Cu，Zn-SOD酶5％、甘油5％、透明质酸1.5％、左旋维生素C1％、维生素E 5％、尿素1％，丙二醇0.5％，余量为去离子水。
对照组1为将H47A+G86W双突变Cu，Zn-SOD酶替换为野生型Cu，Zn-SOD酶。
对照组2为不添加Cu，Zn-SOD酶。
测试人群：选择平均年龄为40±2的健康志愿者60名，随机分为三组，每组20人，分别早晚于面部涂抹上述样品，受试者在实验期间在实验部位不能涂抹任何其它化妆品。
测试方法：
采用水分测试仪(CorneometerCM825)测试皮肤水分含量的变化，评估三种样品对肌肤的保湿程度；用皮肤弹性测试仪(Cutometer dual MPA580)测定皮肤弹性数值的变化。结果如下表2(三次测量平均值)：
表2
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
SEQUENCE LISTING
<110> 深圳市不二生物科技有限公司
<120> 一种突变的超氧化物歧化酶及在制备抗衰老产品中的应用
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 154
<212> PRT
<213> 人工序列
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Claims (7)
1.一种突变的Cu，Zn-SOD酶，所述突变的Cu，Zn-SOD酶的氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的突变的Cu，Zn-SOD酶在用于制备抗衰老化妆品中的应用。
3.如权利要求2所述的应用，其中所述化妆品为精华液或面膜。
4.如权利要求3所述的应用，其中所述精华液包括以下质量百分比的组分：
如权利要求1所述的突变的Cu，Zn-SOD酶2-5％、甘油5-8％、透明质酸0.01-2％、左旋维生素C 0.01-2％、维生素E 2-5％、尿素1-2％，丙二醇0.5-2%，余量为去离子水。
5.如权利要求3所述的应用，其中所述面膜的制备方法为利用权利要求4中所述精华液浸泡面膜材料或添加于面膜材料中。
6.一种抗衰老产品，其特征在于，所述产品为精华液或面膜，所述产品的活性成分包括如权利要求1所述的突变的Cu，Zn-SOD酶。
7.如权利要求6所述的抗衰老产品，其特征在于，所述产品为纳米面膜。
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