CN116042551B - 一种高活性超氧化物歧化酶及其制备方法和在制备抗氧产品中的应用 - Google Patents
一种高活性超氧化物歧化酶及其制备方法和在制备抗氧产品中的应用 Download PDFInfo
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Abstract
一种高活性超氧化物歧化酶(SOD)及其制备方法和在制备抗氧产品中的应用,涉及生物工程技术领域;包括以下步骤:(1)根据目的SOD酶的氨基酸序列,按照毕赤酵母菌密码子优化,人工合成cDNA;所述cDNA的核苷酸序列如SEQ ID NO:3所示;(2)将所述cDNA序列克隆至pPICZaA质粒中,构建重组载体;(3)将所述重组载体转入毕赤酵母菌中,培养并诱导表达,得到高活性超氧化物歧化酶。本SOD容易被皮肤及身体吸收利用。既保留了把自由基转化为水和氧气的第一级抗氧化物的高活性,又实现了耐热性强、稳定性高的特点。在化妆品、保健食品等产品中可保证持久、有效清除自由基对机体造成的损害,延缓机体衰老,在制备抗氧产品的领域中具有良好的应用前景。
Description
技术领域
本发明属于生物工程技术领域,具体涉及表达SOD酶的重组芽孢杆菌及其构建方法和其在制备美白化妆品中的应用。
背景技术
超氧化物歧化酶(superoxide dismutase,SOD)可催化超氧阴离子与氢反应,产生氧气和过氧化氢,是一种重要抗氧化剂,其在保护细胞免受氧自由基的毒性方面发挥着关键作用。SOD常以铜和锌、或锰、铁、或镍作为辅助因子的形式存在。几乎所有真核细胞的细胞内都含有带有铜和锌的超氧化物歧化酶(Cu-Zn-SOD,SOD1);几乎所有的线粒体和许多细菌(如大肠杆菌)均含有结合锰的超氧化物歧化酶(Mn-SOD,SOD2);大肠杆菌和其他一些细菌还含有结合铁的超氧化物歧化酶(Fe-SOD),也有一些细菌只含Fe-SOD或Mn-SOD。人体中SOD有二类,一是位于细胞质和胞外中结合铜离子和锌离子的SOD1,占总SOD的80%以上,另一类是位于线粒体中结合Mn离子的SOD2,人体内不含原核生物的Fe-SOD及少数原始细菌中所含有的Ni-SOD。SeguíJ等人在临床上已证明SOD对结肠炎具有较好疗效。在化妆品中,Vozenin-Brotons MC等人提到可用来清除对皮肤造成损害的自由基,减少肌成纤维细胞的表型逆转来减少纤维化。
胡杨(Populus euphratica)又称胡桐、英雄树、异叶胡杨、异叶杨、水桐、三叶树,是杨柳科杨属胡杨亚属的一种植物,常生长在沙漠中,它耐寒、耐旱、耐盐碱、抗风沙,有很强的生命力。胡杨[CuZn]SOD中包含两段保守His42–Pro65和Ser110-Gly154片段,同时胡杨[CuZn]SOD也具有其他[CuZn]SOD同样保守的络合Cu2+和Zn2+的氨基酸(His-45,His-47,His-62,His-70,His-79,His-119,Asp-82)和两个保守半胱氨酸(Cys-56和Cys-145)。银光委陵菜[CuZn]SOD(PaSOD)第95位含有第3个半胱氨酸(C),而其他的在该位置是苏氨酸(T)或赖氨酸(K),Kumar等人把第95位半胱氨酸突变成丙氨酸(C95A)后,研究表明具有更好的耐热性;但是其耐热性程度还是无法满足实际规模化生产的需要。
巴斯德毕赤酵母(Pichia pastoris)表达系统是近年来发展起来的真核生物基因表达系统之一。该系统可以有效地转录和翻译,准确地完成糖基化、磷酸化、二硫键形成等翻译后加工修饰,能够客观地表达天然蛋白质的结构和功能;可高效表达外源蛋白,具有遗传稳定性。毕赤酵母的醇氧化酶(AOX)基因的启动子具有强诱导性和强启动性,适于外源基因的高水平诱导表达,可高密度发酵等许多优点,应用十分广泛;故研究将巴斯德毕赤酵母应用于高活性超氧化物歧化酶的表达系统中以高效分泌表达高活性的超氧化物歧化酶具有重大的意义。
发明内容
为了克服现有技术的不足,本发明的目的之一在于提供一种高活性超氧化物歧化酶的制备方法,其制备方法简单,易于操作,提高超氧化物歧化酶活性,并具有良好的耐热性,且生产效率高。
本发明的目的之二在于提供高活性超氧化物歧化酶,其具有活性高,耐热性强,稳定性高特点。
本发明的目的之三在于提供一种高活性超氧化物歧化酶的应用。
本发明的目的之一采用如下技术方案实现:
一种高活性超氧化物歧化酶的制备方法,包括以下步骤:
(1)根据目的SOD酶的氨基酸序列,按照毕赤酵母菌密码子优化后,人工合成cDNA;所述cDNA的核苷酸序列如SEQ ID NO:3所示;
(2)将所述cDNA序列克隆至pPICZaA质粒中,构建重组载体;
(3)将所述重组载体转入毕赤酵母菌中,培养并诱导表达,得到高活性超氧化物歧化酶。
进一步地,步骤(1)中,目的SOD酶为氨基酸序列如SEQ ID NO:1所示的SOD酶。
进一步地,所述SOD酶由氨基酸序列如SEQ ID NO:2所示的超氧化物歧化酶定点突变而成。
进一步地,所述定点突变包括:将所述超氧化物歧化酶的第9位的天冬酰胺突变成丝氨酸,第15位的天冬酰胺突变成赖氨酸,第41位的脯氨酸突变成亮氨酸。
进一步地,步骤(3)中,具体操作为:将所述重组载体电转入感受态毕赤酵母菌后,得到重组毕赤酵母菌,培养并诱导表达,得到高活性超氧化物歧化酶。
进一步地,将重组毕赤酵母菌接入YPD培养基中,在28-30℃、200-300rpm条件下摇床培养至OD600=0.8-1.0,按1:100-300的接种量接入BMGY培养基中,在28-30℃、200-300rpm条件下摇床培养至OD600=2.0-6.0,换成转移到含10.5-1.5%甲醛的BMMY培养基,补加0.5-1.5%甲醇至1%/24小时诱导表达,得到高活性超氧化物歧化酶。本发明的目的之二采用如下技术方案实现:
一种高活性超氧化物歧化酶,由所述的高活性超氧化物歧化酶的制备方法所制成。
本发明的目的之三采用如下技术方案实现:
一种高活性超氧化物歧化酶的应用,所述的高活性超氧化物歧化酶在制备抗氧产品中的应用。
相比现有技术,本发明的有益效果在于:
本发明的一种高活性超氧化物歧化酶的制备方法,其制备方法简单,易于操作;按照毕赤酵母菌优化密码子及mRNA高级结构后的人工合成cDNA后,构建得到重组毕赤酵母菌,有效提高超氧化物歧化酶的产率,提高超氧化物歧化酶的活性;超氧化物歧化酶为以超氧化物歧化酶为模板,经多点突变(N9S,N15K,P41L)获得,其耐热性大幅提升,协同重组毕赤酵母菌的表达系统,能高效生产超氧化物歧化酶,提高活性,且超氧化物歧化酶的耐热性强、稳定性高,可长期保藏。
本发明的高活性超氧化物歧化酶,其具有耐热性强,稳定性高,且分子量小,容易被吸收。
本发明的一种高活性超氧化物歧化酶的应用,超氧化物歧化酶的抗氧化性强,能有效去除氧自由基以保护细胞,且分子量小,容易被皮肤吸收利用,在制备抗氧产品的领域中具有良好的应用前景。
附图说明
图1是本发明的实施例3的凝胶电泳分析图;其中,M:蛋白marker(Solarbio,PR1920);泳道1-4:48h表达上清;泳道5-8:72h表达上清;泳道9:纯化产物。
图2是本发明的PmPeSOD2-1在不同温度处理下的生物活性分析图。
图3是本发明的PmPeSOD2-1在不同浓度尿素下的活性分析图。
具体实施方式
下面,结合具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
在NCBI数据库中搜索得到12条gene与SOD相关,均来自幼发拉底胡杨的基因组测序Gnomon预测分析结果,其中7条[CuZn]SOD预测基因,3条[Fe]SOD和2条[Mn]SOD基因序列;7条[CuZn]SOD预测基因中3条来自叶绿体,其余4条基因包含测序得到的6个转录本和人及其他耐热植物CuZnSOD对比。
clastalX的对比结果显示:XP_011023491.1和XP_011023492.1多肽序列完全相同,来源于同一基因座(LOC105124956);XP_011032547.1和XP_011032548.1多肽序列也完全相同,来自于同一基因座(LOC105131320);而XP_011034389.1和XP_011021470.1两条[CuZn]SOD2-like与其他两条序列并不属于一类,差异较大。对比结果还显示胡杨[CuZn]SOD也包含两段保守His42–Pro65和Ser110-Gly154片段,同时胡杨[CuZn]SOD也具有其他[CuZn]SOD同样保守的络合Cu2+和Zn2+的氨基酸(His-45,His-47,His-62,His-70,His-79,His-119,Asp-82)和两个保守半胱氨酸(Cys-56和Cys-145)。
通过以上对比分析,我们挑选假定的PeSOD2转录变异体1(XP_011032547.1)的多肽序列为蓝本(命名为PeSOD2-1),以其为模板,氨基酸如SEQ ID NO:2所示,研究SOD高级结构和保守残基及其他植物耐热[CuZn]SOD序列,对其进行定点突变(N9S,N15K,P41L);所述定点突变具体为:将所述超氧化物歧化酶的第9位的天冬酰胺(Asn)突变成丝氨酸(Ser),第15位的天冬酰胺(Asn)突变成赖氨酸(Lys),第41位的脯氨酸(Pro)突变成亮氨酸(Leu),命名为mPeSOD2-1,其氨基酸序列如SEQ ID NO:1所示。
用SOPMA分析显示mPeSOD2-1含有3.95%α螺旋(h),32.89%的β片层(extendedstrand,e),6.58%的β-turn(t),56.58%随机卷曲(c)。
实施例2
构建重组毕赤酵母:
取实施例1的超氧化物歧化酶mPeSOD2-1,按照毕赤酵母(Pichia pastoris)优化密码子及mRNA高级结构后人工合成cDNA序列,获得的cDNA序列如SEQ ID NO:3所示,用XhoI和NotI双酶切直接亚克隆入pPICZαA中,构建重组载体;优选地,mPeSOD2-1多肽重组载体C端上融合His-tag标签,以便于后续纯化和鉴定。
具体为:
1、挑取划线单克隆于2ml YPD,30℃×230rpm×12-16h;
2、取10ul过夜菌加入50ml YPD中,30℃×230rpm摇至OD600=1.0-1.3,3、倒入50mlEP管,4000rpm离心5min,弃净上清
4、加入50ml冰水浴超纯水,轻轻混匀;
5、4℃×4000rpm×5min,弃净上清,加入30-50ml冰水浴超纯水,轻混匀,冰浴5min;
6、4℃×4000rpm×5min,弃净上清,加入2ml冰水浴1M山梨醇,Mix;
7、4℃×4000rpm×5min,吸弃上清,加入80-100ul 1M山梨醇,用中枪头混匀菌块,吸取80ul至冰浴1.5ml EP,放冰上当天使用。
8、取<10ul高质量线性化变性质粒DNA 10ug加入刚制备感受态中,混匀,移入冰浴0.2cm电转杯中,冰浴5-10min,1500V,25μF,200Ω电转化后立即加入1ml 1M山梨醇,30℃×静置1-3h,涂100ug/ml Zeocin YPDS板,30℃培养3-5天待克隆长出。
9、酵母高拷贝转化子的筛选:挑取100μg/mL Zeocin YPDS平板上单菌落,在高浓度Zeocin YPDS平板(500、1000μg/mL)上点种培养,在该平板上生长速度及菌落大小与100μg/mL Zeocin YPDS平板上相同的转化子有可能含高拷贝外源基因,用于进一步表达实验。
实施例3
1、诱导重组毕赤酵母的表达:
挑取实施例2的筛选出的高copy重组的单克隆,将重组毕赤酵母菌接入YPD培养基中,在28-30℃、200-300rpm条件下摇床培养至OD600=0.8-1.0,按1:100-300的接种量接入BMGY培养基中,在28-30℃、200-300rpm条件下摇床培养至OD600=2.0-6.0,换成含1%甲醛的BMMY培养基,补加甲醇至1%/24小时。分别在0h、24h、48h、3天、4天、5天和第6天各收菌500μl。发酵菌液在室温12000rpm离心2min,取上清40μl加入10ul 5×SDS-PAGE上样缓冲液,混均,100℃加热5min,置冰上,10000rpm离心2min,各取20μl上清上样用SDS-PAGE凝胶电泳分析表达情况,结果如图1所示。
2、mPeSOD2-1在毕赤酵母中诱导表达产物纯化:
在实施例3中采用甲醇诱导表达上清,取1ml加入200ul PBS洗涤的Ni-resin(碧云天生物科技,P2218),室温用混悬仪8rpm孵育10min,凝胶用PBS洗涤3次,用5mM咪唑洗涤去除杂蛋白,用30mM咪唑洗脱目的蛋白,洗脱液加SDS-PAGE上样缓冲液,混合均匀,100℃加热5min,10000rpm离心2min,取15μl上清上样用SDS-PAGE凝胶电泳分析纯化情况,结果如图3(泳道9)所示,得到纯化后的目的带(理论分子量约17kDa)。目的蛋白用0.1M磷酸钾(pH7.5)或PBS透析即可得到Ni纯化后的mPeSOD2-1蛋白。
性能测试
1、不同温度处理对mPeSOD2-1生物活性影响:诱导表达72h上清液,按1:50稀释,分别在60、70℃、80℃、90℃、100℃不同温度处理10min后放冰上,检测各溶液中[CnZn]mPeSOD2-1活性。
结果如图2A所示,100℃以内孵育10min对[CnZn]mPeSOD2-1活性无显著影响;其中,系列1-4:代表4个不同的克隆发酵样本。
在60、70℃、80℃、90℃、100℃处理分别加热20、40、60、80、100min;结果如图2B所示,在60、70、80℃处理100min或90℃处理60min以内,对[CnZn]mPeSOD2-1活性无显著影响。
另外,干粉或溶液状态在4℃以下至少保存两年酶活维持在95%以上,冻融几乎不影响活性。
2、不同浓度尿素对mPeSOD2-1活性影响:
取酵母表达mPeSOD2-1上清,按1:50用PBS稀释,稀释后的酶液分成7管,加入0、0.1、0.2、0.3、0.5、1、2、4M的尿素,37℃孵育1h,测[CnZn]mPeSOD2-1活性。
结果如图3所示,mPeSOD2-1对低浓度(<0.5M)尿素有较强的耐受性,2M以上的尿素才对其活性影响较大,使其大部分变性失活,证明本发明的mPeSOD2-1具有较高的稳定性。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (7)
1.一种高活性超氧化物歧化酶的制备方法,其特征在于:包括以下步骤:
(1)根据目的SOD酶的氨基酸序列,按照毕赤酵母菌密码子优化后,人工合成cDNA;所述cDNA的核苷酸序列如SEQ ID NO:3所示;
(2)将所述cDNA序列克隆至pPICZαA质粒中,构建重组载体;
(3)将所述重组载体转入毕赤酵母菌中,培养并诱导表达,得到高活性超氧化物歧化酶。
2.如权利要求1所述的高活性超氧化物歧化酶的制备方法,其特征在于:步骤(1)中,目的SOD酶为氨基酸序列如SEQ ID NO:1所示的SOD酶。
3.如权利要求2所述的高活性超氧化物歧化酶的制备方法,其特征在于:所述SOD酶由氨基酸序列如SEQ ID NO:2所示的超氧化物歧化酶定点突变而成。
4.如权利要求3所述的高活性超氧化物歧化酶的制备方法,其特征在于:所述定点突变包括:将所述超氧化物歧化酶的第9位的天冬酰胺突变成丝氨酸,第15位的天冬酰胺突变成赖氨酸,第41位的脯氨酸突变成亮氨酸。
5.如权利要求1所述的高活性超氧化物歧化酶的制备方法,其特征在于,步骤(3)中,具体操作为:将所述重组载体电转入感受态毕赤酵母菌后,得到重组毕赤酵母菌,培养并诱导表达,得到高活性超氧化物歧化酶。
6.如权利要求5所述的高活性超氧化物歧化酶的制备方法,其特征在于,诱导表达步骤包括:
将重组毕赤酵母菌接入YPD培养基中,在28-30℃、200-300rpm条件下摇床培养至OD600=0.8-1.0,按1:100-300的接种量接入BMGY培养基中,在28-30℃、200-300rpm条件下摇床培养至OD600=2.0-6.0,转移到含0.5-1.5%甲醛的BMMY培养基,补加0.5-1.5%甲醇诱导表达,得到高活性超氧化物歧化酶。
7.一种高活性超氧化物歧化酶,其特征在于:由权利要求1-6任一项所述的高活性超氧化物歧化酶的制备方法所制成。
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