CN116121214B - Sod突变体及其在重组芽孢杆菌的表达方法和在制备美白化妆品中的应用 - Google Patents
Sod突变体及其在重组芽孢杆菌的表达方法和在制备美白化妆品中的应用 Download PDFInfo
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Abstract
SOD突变体及其在重组芽孢杆菌的表达方法和在制备美白化妆品中的应用；包括以下步骤：(1)根据目的SOD突变体的氨基酸序列，按照枯草芽孢杆菌优化密码子及mRNA高级结构后，人工合成cDNA；(2)构建重组载体；(3)将所述重组载体转入枯草芽孢杆菌中，培养得到重组芽孢杆菌。SOD突变体为以超氧化物歧化酶为模板，经多点突变获得，其耐热性大幅提升，协同重组芽孢杆菌的表达***，能高效生产SOD突变体，且SOD突变体的耐热性强、稳定性高，可长期保藏。SOD对皮肤酪氨酸酶活性有显著抑制作用，可美白肌肤、提亮肤色，在受紫外线照射时保护皮肤，减少皮肤发红，在制备防晒美白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SOD突变体及其在重组芽孢杆菌的表达方法和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。
枯草芽孢杆菌(Bacillus subtilis)，是芽孢杆菌属的一种，革兰氏阳性菌，可形成内生抗逆芽孢；枯草芽孢杆菌菌体生长过程中产生的枯草菌素等活性物质，这些活性物质对致病菌或内源性感染的条件致病菌有明显的抑制作用；同时芽孢杆菌具有较高的蛋白合成与分泌能力，易于胞外分泌目的蛋白，可直接在胞外培养基中收集生产的目的蛋白；且芽孢杆菌对人及其它动物没有致病性；故将枯草芽孢杆菌应用于SOD突变体的表达***中具有重要的意义。
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SOD突变体在重组芽孢杆菌的表达方法，其方法简单，易于操作，有效提高SOD突变体的产率，并使SOD突变体具有良好的稳定性。
本发明的目的之二在于提供一种SOD突变体，OD突变体具有耐热性强，稳定性高，渗透能力强的特点。
本发明的目的之三在于提供一种SOD突变体在制备美白化妆品中的应用。
本发明的目的之一采用如下技术方案实现：
一种SOD突变体在重组芽孢杆菌的表达方法，包括以下步骤：
(1)根据目的SOD突变体的氨基酸序列，按照枯草芽孢杆菌优化密码子及mRNA高级结构后，人工合成cDNA；所述cDNA的核苷酸序列如SEQ IDNO:4或SEQ ID NO:5所示；
(2)将所述cDNA序列克隆至pHT1469质粒中，构建重组载体；
(3)将所述重组载体转入枯草芽孢杆菌中，诱导表达，得到SOD突变体。
进一步地，步骤(1)中，目的SOD突变体包括氨基酸序列如SEQ ID NO:1所示的SOD突变体及其N端或C端修饰得到的SOD衍生突变体。
进一步地，所述SOD突变体由氨基酸序列如SEQ ID NO:2所示的超氧化物歧化酶定点突变而成；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，所述SOD衍生突变体由所述SOD突变体N端或C端去掉1-9个氨基酸后修饰多肽制得。
进一步地，所述SOD衍生突变体的氨基酸序列如SEQ ID NO:3所示。
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枯草芽孢杆菌后，培养得到表达SOD突变体的重组芽孢杆菌，诱导表达，得到SOD突变体。
进一步地，诱导表达的操作为：将重组芽孢杆菌接入含15-25μg/ml氯霉素的LB培养基中，在28-32℃、200-300rpm条件下摇床培养至OD600＝0.8-1.0，按1:80-120接入含15-25μg/ml氯霉素的诱导培养基中，在28-32℃、200-300rpm条件下摇床培养至OD600＝0.8-1.0，加入0.8-1.2mM IPTG诱导表达，得到SOD突变体。
进一步地，所述诱导培养基包括以下组分：
胰蛋白胨1.4-1.8wt％，酵母提取物0.3-0.7wt％，葡糖糖1.5-2.5wt％，Tween400.2-0.4wt％，无机盐90-120mM；
其中，按摩尔百分比计，所述无机盐包括：FeCl3 48-52％，CaCl2 18-22％，
MnCl2 8-12％，ZnSO4 8-12％，CoCl2 1-3％，CuCl2 1-3％，NiCl2 1-3％，Na2MoO41-3％，H3BO31-3％。
本发明的目的之二采用如下技术方案实现：
一种SOD突变体，由所述的SOD突变体在重组芽孢杆菌的表达方法所得。
本发明的目的之三采用如下技术方案实现：
一种SOD突变体的应用，所述SOD突变体在重组芽孢杆菌的表达方法得到的SOD突变体在制备美白化妆品中的应用。
相比现有技术，本发明的有益效果在于：
本发明的一种SOD突变体在重组芽孢杆菌的表达方法，其方法简单，易于操作；按照枯草芽孢杆菌优化密码子及mRNA高级结构后的人工合成cDNA后，与枯草芽孢杆菌构建得到重组芽孢杆菌，有效提高SOD突变体的产率，保持SOD突变体的完整活性；SOD突变体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，协同重组芽孢杆菌的表达***，能高效生产SOD突变体，且SOD突变体的耐热性强、稳定性高，可长期保藏。
本发明的一种表达SOD突变体，其具有耐热性强，稳定性高，渗透能力强的特点，且分子量小，容易被吸收。
本发明的一种SOD突变体在制备美白化妆品中的应用。SOD突变体的抗氧化性强，能有效去除氧自由基以保护细胞，且渗透能力强，分子量小，容易被皮肤吸收利用，在制备美白化妆品的领域中具有良好的应用前景。
附图说明
图1A是本发明的实施例3中PmPeSOD2-1在枯草芽孢杆菌中表达纯化的SDS-PAGE凝胶电泳图；其中，M:蛋白质分子量标准(上海碧云天生物技术有限公司，P0075)；泳道1：未诱导菌培养上清液0h；泳道2：诱导12h菌培养上清液；泳道3：诱导24h菌培养上清液；泳道4：诱导36h菌培养上清液；泳道5：诱导48h菌培养上清液；泳道6：诱导72h菌培养上清液；泳道7：诱导96h菌培养上清液；泳道8：诱导第5天菌培养上清液；泳道9：诱导第6天菌培养上清液。
图1B是本发明的实施例3中诱导72h培养上清液用硫酸铵沉淀纯化后的SDS-PAGE分析图；其中，M:蛋白质分子量标准(上海碧云天生物技术有限公司，P0075)；泳道b1：40％硫酸铵沉淀；泳道b2：45％硫酸铵沉淀；泳道b3：50％硫酸铵沉淀；泳道b4：60％硫酸铵沉淀。
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
将实施例1的mPeSOD2-1在N端去掉3个氨基酸后连接多肽进行修饰，得到的多肽记为PmPeSOD2-1，其氨基酸序列如SEQ ID NO:3所示。本实施例修饰多肽后，其渗透能力强，分子量小，容易被吸收。
实施例3
构建并诱导表达SOD突变体的重组芽孢杆菌：
取实施例2的超氧化物歧化酶突变体PmPeSOD2-1，按照枯草芽孢杆菌(B.subtilis)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 IDNO:4所示，用BamHI和XbaI双酶切直接亚克隆入pHT1469(MoBiTec Gmbh,PBS018)中，构建重组载体；优选地，PmPeSOD2-1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：
制备电转感受态的枯草芽孢杆菌WB800N(MoBiTec Gmbh,PBS022)：挑选新鲜LB平板单菌落接种于3ml LB培养基中，30℃培养过夜。取过夜培养后的菌种接种至50ml SLB培养基(LB培养液+0.5M山梨醇，pH值为7.2)中，控制接种量为OD＝0.19-0.2之间，30℃，250rpm下培养至OD600＝0.8-1.0(约4h)。将全部菌液冰水浴10min，然后下5000rpm，8min，4℃下离心收集菌体。用40ml预冷超纯水或1mM Hepes液(pH 7.0)，4℃，5000rpm，8min下洗涤菌体3次。再将菌体用5ml PM洗涤，加1ml PM重悬，分装100μl/管，-80冻存。
取一支WB800N感受态加3μl重组载体(～200ng)/100μl感受态，混均移入预冷1mm电转杯中，冰浴5min；2000V,25uF,200Ω电击一次，立即加入1ml RM复苏培养基(LB培养液+0.5M山梨醇+0.38M甘露醇，pH值为7.2)混合，30℃静置复苏3h，5000rpm离心3min，吸弃上清剩余约50μl，吹打均匀全涂在含氯霉素(5-10μg/ml)LB平板上，倒扣30℃培养过夜，得到重组芽孢杆菌。
挑取单克隆，接入1ml含20μg/ml氯霉素LB培养基中，放30℃摇床250rpm摇至OD600＝0.8-1.0，按1:100接入15ml 2YT培养基中(含20μg/ml氯霉素)，30℃，250rpm摇至OD600＝0.8-1.0，加入1mM IPTG诱导表达，分别在0h、12h、24h、36h、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1A所示。
参照图1A，与未诱导样本相比较，诱导的菌体中在理论分子量17.5kDa处有明显的目的带。诱导48h后有较明显的目的蛋白表达，但其后随诱导时间延长，目的蛋白分泌并没有增多，推测可能与培养基营养有关。
取诱导分泌表达72h的离心上清，分别加入终浓度40％、45％、50％、60％的硫酸铵，混均，冰浴1h，12000rpm离心15min，沉淀用超滤管(Amicon Ultra-4，10kDa)洗涤浓缩，用SDS-PAGE分析(图1B)。参照图1B，用饱和硫酸铵在滴加至终浓度40％时即有明显目的带沉淀富集出来。
实施例4
取实施例1的超氧化物歧化酶突变体mPeSOD2-1，按照枯草芽孢杆菌(B.subtilis)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:5所示，其在枯草芽孢杆菌(B.subtilis)中诱导表达过程与实施例3相同。
实施例5
枯草芽孢杆菌分泌SOD突变体表达的影响条件
不同培养基或者培养基中组分比例对宿主菌生长和蛋白合成尤为重要，Mg离子对大多数细菌高密度生长及蛋白合成是必要的，而Ca2+存在可协助葡萄糖进入细菌胞内，Tween 40等非离子去污剂可以通过干扰细胞壁的通透性来促芽孢杆菌分泌表达，一定浓度Gly(甘氨酸)也可以促进宿主菌的蛋白合成和胞外分泌，不同时间点补加碳源有利于目的蛋白合成，基于次设计不同培养和诱导分泌表达条件。
从实施例3的重组芽孢杆菌中挑取单克隆，接入1ml含20μg/ml氯霉素LB培养基中，放30℃摇床250rpm摇至OD600＝0.8，按1:100接入5ml诱导培养基中(含20μg/ml氯霉素)；30℃，250rpm下摇至OD600＝0.8，各加入1mM IPTG诱导表达，在24h、48h和72h取上清液，测OD值和按照国标GB/T 5009.171-2003中所述邻苯三酚法进行测试，测分泌上清中PmPeSOD2-1活性。其中，调水的每分钟ΔA’值＝0.070，用个样本的每分钟ΔA值来表征酶活性。诱导培养基的成分和结果如表1所示。
表1
注：1000×M无机盐包括：50mM FeCl3，20mM CaCl2，10mM MnCl2，10mMZnSO4，2mMCoCl2，2mM CuCl2，2mM NiCl2，2mM Na2MoO4，2mM H3BO3。
从表1可以看出，用M2培养基(胰蛋白胨1.6％，酵母提取物0.5％，葡糖糖2％)+无机盐+0.3％ Tween诱导枯草芽孢杆菌表达时ΔA最小，代表上清液中SOD突变体活性最高，在发酵72h时上清原液约160U/ml。
经测试，SOD突变体PmPeSOD2-1和mPeSOD2-1还具有以下性能：
1、耐热性
将SOD突变体分别90℃保温120分钟和100℃保温40分钟，该复合肽保持完整活性；说明其具有显著的耐热性。
2、稳定性
SOD突变体在pH值为4.0-11.0范围内酶活稳定，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。
3、渗透性
SOD突变体PmPeSOD2-1的渗透能力强，且可在皮肤尤其是损伤部位自行分解为两独立活性肽，分子量小，容易被吸收。
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 (10)
1.一种超氧化物歧化酶（SOD）突变体在重组芽孢杆菌的表达方法，其特征在于：包括以下步骤：
（1）根据目的SOD突变体的氨基酸序列，按照枯草芽孢杆菌优化密码子及mRNA高级结构后，人工合成cDNA；所述cDNA的核苷酸序列如SEQ ID NO:4所示；
（2）将所述cDNA序列克隆至pHT1469质粒中，构建重组载体；
（3）将所述重组载体转入枯草芽孢杆菌中，诱导表达，得到SOD突变体。
2.如权利要求1所述的SOD突变体在重组芽孢杆菌的表达方法，其特征在于：步骤（1）中，目的SOD突变体是氨基酸序列如SEQ ID NO:1所示的SOD突变体及其N端修饰得到的SOD衍生突变体。
3.如权利要求2所述的SOD突变体在重组芽孢杆菌的表达方法，其特征在于：所述SOD突变体由氨基酸序列如SEQ ID NO:2所示的超氧化物歧化酶定点突变而成；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。
4.如权利要求2所述的SOD突变体在重组芽孢杆菌的表达方法，其特征在于：所述SOD衍生突变体由所述SOD突变体N端去掉3个氨基酸后修饰多肽制得。
5.如权利要求4所述的SOD突变体在重组芽孢杆菌的表达方法，其特征在于：所述SOD衍生突变体的氨基酸序列如SEQ ID NO:3所示。
6.如权利要求1所述的SOD突变体在重组芽孢杆菌的表达方法，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枯草芽孢杆菌后，培养得到表达SOD突变体的重组芽孢杆菌，诱导表达，得到SOD突变体。
7.如权利要求6所述的SOD突变体在重组芽孢杆菌的表达方法，其特征在于，诱导表达的操作为：将重组芽孢杆菌接入含15-25µg/ml氯霉素的LB培养基中，在28-32℃、200-300rpm条件下摇床培养至OD600=0.8-1.0，按1:80-120接入含15-25µg/ml氯霉素的诱导培养基中，在28-32℃、200-300rpm条件下摇床培养至OD600=0.8-1.0，加入0.8-1.2mM IPTG诱导表达，得到SOD突变体。
8.如权利要求7所述的SOD突变体在重组芽孢杆菌的表达方法，其特征在于，所述诱导培养基包括以下组分：
胰蛋白胨1.4-1.8wt%，酵母提取物0.3-0.7wt%，葡糖糖1.5-2.5wt%，Tween40 0.2-0.4wt%，无机盐 90-120mM；
其中，按摩尔百分比计，所述无机盐包括：FeCl3 48-52%，CaCl2 18-22%，
MnCl2 8-12%，ZnSO4 8-12%，CoCl2 1-3%，CuCl2 1-3%， NiCl2 1-3%，Na2MoO41-3%，H3BO31-3%。
9.一种SOD突变体，其特征在于：由权利要求1-8任一项所述的SOD突变体在重组芽孢杆菌的表达方法所得。
10.一种SOD突变体的应用，其特征在于：权利要求9所述SOD突变体在制备美白化妆品中的应用。
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