CN104371984A - 一组可提高sod抗逆性和水溶液中稳定性的氨基酸序列及其应用 - Google Patents
一组可提高sod抗逆性和水溶液中稳定性的氨基酸序列及其应用 Download PDFInfo
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- CN104371984A CN104371984A CN201410674001.4A CN201410674001A CN104371984A CN 104371984 A CN104371984 A CN 104371984A CN 201410674001 A CN201410674001 A CN 201410674001A CN 104371984 A CN104371984 A CN 104371984A
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Abstract
本发明涉及一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列及其应用，它是一组来源于 Geobacillus 属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列。这组氨基酸序列具有以下特征：1）位于Fe/Mn-SOD蛋白的sodA功能域之前，序列长度为101-344个氨基酸，功能未知；2）其相互之间具有85-100%的同源性；3）均包含1或2个特殊的共有重复序列Geo-N-repeat。这组氨基酸序列对 Geobacillus 属的Fe/Mn-SOD的抗逆性和水溶液中稳定性具有决定性作用，可广泛应用于其它SOD（特别是传统工业用SOD）的改造，显著提高其抗逆性和水溶液中稳定性。
Description
技术领域
本发明涉及一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列及其应用。
背景技术
超氧化物歧化酶（surperoxide dismutase，EC.1.5.1.1，SOD）是一类金属酶，该酶广泛存在于动物、植物和微生物等多种生物体内。1938年由Mann和Keilin从牛红细胞中分离得到。1969年Mecord和Fridovich发现该蛋白质能催化清除超氧阴离子自由基（O2-）的反应，是一种重要的氧自由基清除剂，可平衡机体的氧自由基，从而避免当机体内超氧阴离子自由基浓度过高时引起的不良反应。按其结合的金属离子可分为Fe-SOD，Mn-SOD，Cu/ZnSOD三种。SOD具有抗炎、抗病毒、抗辐射、抗衰老等作用，其也在病毒性疾病、自身免疫性疾病、心肌缺血和缺血再灌流综合症、老年性白内障、心血管疾病、辐射病、癌症的治疗预防以及人类长寿等领域中起到重要作用。
天然SOD在工业生产和食品药品添加过程中有半衰期短，遇变性剂、抑制剂易失活等不利因素。SOD的稳定性是决定其能否商业化的一个重要条件。因此，对SOD进行修饰改造，改变分子的物理化学性质，增加其对高温、极端pH、变性剂、清洁剂、抑制剂等的抗逆性及在水溶液中的稳定性，克服上述不利因素就显得十分必要。目前国内外已有很多的研究，首先是化学修饰，如采用聚乙二醇、右旋糖酐、月桂酰氯、多糖物质等对SOD的氨基和胍基进行化学修饰以提高其稳定性并仍能保留最大活性；其次是基因重组，国际生物学界采用的基因重组法制取人源SOD是SOD制备的最前沿技术，具有高安全、高活性、高稳定性等优势；最后是SOD的模拟化合物，其具备更低的分子量和更高的稳定性，且获取和制备比天然SOD简单。本专利涉及一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列，为实现SOD的稳定性改造提供了一种全新的思路和方法，该方法操作简便、可行性强、适应性好，可实现传统工业用SOD的升级换代，更有利于SOD酶制剂在成分复杂的食品、药品和化妆品中的添加，并保持长期稳定，延长货架期，具有重要的应用价值和前景。
发明内容
本发明的一个目的是提供一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，这13个N端氨基酸序列具有以下特征：
（1）位于Fe/Mn-SOD蛋白的sodA功能域之前，序列长度为101-344个氨基酸，不能独立行使SOD的功能；
（2）其相互之间具有85-100%的同源性；
（3）都包含特殊的共有重复序列Geo-N-repeat，其中9个N端序列含有2个重复序列，另外4个N端序列含有1个重复序列。
本发明的另一目的是提供一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，这一组N端氨基酸序列对Geobacillus属的Fe/Mn-SOD的抗逆性和水溶液中稳定性具有决定性作用，除去这一组N端氨基酸序列后的Geobacillus属的Fe/Mn-SOD的抗逆性和水溶液中稳定性大幅降低（实施例1所示）。
本发明的另一目的是提供一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，这组N端氨基酸序列可广泛应用于其它SOD（不仅限于SOD），特别是传统工业用SOD的抗逆性和水溶液中稳定性改造。通过一定方法，将这段N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端，可以显著提高SOD的抗逆性、水溶液中及冻干粉的稳定性（如实施例1、2所示）。
本发明的另一目的是提供一种能表达重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组质粒。
本发明的再一目的是提供一种能产生上述重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组菌。
本发明的再一目的是提供了SEQ ID NO:1-13氨基酸序列在提高SOD的抗逆性、提高SOD在水溶液中稳定性方面的应用。
为了实现上述目的，本发明采用如下技术方案：
本发明提供一组来源于 Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，如SEQ ID NO:1-13所示。
上述一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，其特征在于：（1）位于Fe/Mn-SOD蛋白的sodA功能域之前，序列长度为101-344个氨基酸；（2）其相互之间具有85-100%的同源性；（3）都包含特殊的共有重复序列Geo-N-repeat，其中SEQ ID NO:1-9的9个N端序列都含有2个重复序列，SEQ ID NO:10-13的4个N端序列都含有1个重复序列。
上述一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，其具有与SEQ ID NO:1-13所示的氨基酸序列至少70%的同源性。
上述一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，其具有SEQ ID NO:1-13所示的氨基酸序列中缺失、替换、或插入的多个氨基酸序列为2-100个。
上述一组来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，其特征在于，这组N端氨基酸序列可广泛应用于其它SOD（不仅限于SOD），特别是传统工业用SOD的抗逆性和水溶液中稳定性改造。通过一定方法，将这段N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端，可以显著提高SOD的抗逆性和水溶液中稳定性。
本发明提供的SEQ ID NO:1-13氨基酸序列如下：
Geobacillus thermodenitrificans NG80-2SEQ ID NO:1
MDDQTLFAQYAAEVNEWGEQVKQVLELRGASIDGASTLLQFIAEHDGKWTEEAVRELTRLVDDVYAAALRHYAIEAAEWGKQVEHALSMRGAAEDIGLSSLLARIEEHGDEWTEEEIHELQLLVDDVYARAIRLVEPLSDGQEEDLTRQEEVSALPEQEGGNREQMSKGTERSGEHKGDSEQEPVVAAERAEPFIASSTDSPDGEQLHEGDTMDEEWRHNADMTDKERLPEEGVTDGERQRAVS
Geobacillus thermoleovorans CCB_US3_UF5SEQ ID NO:2
MRLDKETVWCSRRFAKAACLFQKKGGADPKREYVLGQPDFFERFIYGSFADPLQGSITAILFFLAVFSYDDCPRSSMYERPGIWVNADKNDFPMTAYDMANKTGGGFYQMDDQVLFAQYAARVNEWGNQVKETLALRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAADREEEATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSPTDAADGEPDQADNVTDGKRRWVDADDGGEPRQQVAPGR
Geobacillus sp. G11MC16SEQ ID NO:3
MDDQALFAQYAAEVNEWGEQVKQVLELRGASIDGASTLLQFIAEHDGEWTEEAVRELTRLVDDVYAAALRHYAIEAAEWGKQVEHALSMRGAAEDIGLSSLLARIEEHGDEWTEEEIHELQLLVDDVYARAIRLVEPLSDGQEEDLTRQEEVSALPEQEGGNGEQMSEGTERSGEHKGDSEQEPVVAAERAEPFIASSTDSPDGEQLHEGDTMDEEWRHNADMTDKERLTEEGVTDGERQRAVS
Geobacillus sp. C56-T3SEQ ID NO:4
MDDQVLFAQYAARVNEWGNQVKETLALRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAAEREEEATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSLPDIADGEPGQADNVTDGKRRWVDADDGGEPRQQAAPGR
Geobacillus sp. Y412MC52SEQ ID NO:5
MDDQVLFAQYAARVNEWGNQVKETLALRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAAEREEEATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSLPDIADGEPGQADNVTDGKRRWVDADDGGEPRQQVAPGR
Geobacillus sp. GHH01SEQ ID NO:6
MDDQVLFAQYAARVNEWGNQVKETLALRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAAEREEEATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSLPDIADGEPGQADNVTDGKRRWVDADDGGEPRQQAAPGR
Geobacillus sp. Y412MC61SEQ ID NO:7
MDDQVLFAQYAARVNEWGNQVKETLALRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAAEREEEATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSLPDIADGEPGQADNVTDGKRRWVDADDGGEPRQQVAPGR
Geobacillus kaustophilus HTA426SEQ ID NO:8
MRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAADREEEATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSPTDAADGEPDQADNVTDGKRRWVDADDGGEPRQQVAPGR
Geobacillus sp. POT5SEQ ID NO:9
LRGASTDGASSLLEFIAEHDGEWTEEAVRELQRLADDVYVGALRQYVMEAAAWGRQVEQALSARRMAEDVGLSSLLAYIDGHGDEWTEEAIYELQRLVDDVYTRAVRLADSSAADREEGATQEQVEGESVSPELESENKENEDGWLDTSGTAERVEDAKEPAFMAELSDSPTDAADGEPDQVDNVTDGKRRWVDADDGGEPRQQVAPGR
Geobacillus sp. WCH70SEQ ID NO:10
MNEQERIQQYVAEVKEWGKQVEQILLQRGEDGGDCRVDSLLSYIEHHGDAWTEDAIYELQRMVDEVYEKALVFQQNGQSAIRQEESGTEERQQTSIGQEENGIEERQQTAVRQEESGTEERQQTASEQEEESEAEERQQTYVAAGR
Geobacillus thermoglucosidans TNO-09.020SEQ ID NO:1 1
MSEQELFQRYVKQVSEWGAQVGQMLPRRDDGTVHHDIAALLSYIDRHDGEWTETEIYDLQRMADAVYEKAAAVSANGTLADESRETSEEEARRQTYITAGR
Geobacillus thermoglucosidasius C56-YS93SEQ ID NO:12
MSEQELFQRYVKQVSEWGAQVGQMLPRRDDGTVHHDIAALLSYIDRHDGEWTETEIYDLQRMADAVYEKAAAVSANGTLADESRETSEEEARRQTYITAGR
Geobacillus sp. Y4.1MC1SEQ ID NO:13
MSEQELFQRYVKQVSKWGAQVGQMLPRRDDGTVHRDIAALLSYIDRHDGEWTETEIYDLQRMADAVYEKAAAVSANGTLADESRETSEEEARRQTYITAGR
一种表达上述的超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组质粒，该质粒至少包括SEQ ID NO:1-13所示的基因。
上述表达重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组质粒的载体为pET-28a(+)（实验室常用载体）。
一种产生上述的重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组菌，该重组菌导入了超氧化物歧化酶。
上述产生重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组菌为大肠杆菌。
上述产生重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的重组菌的大肠杆菌为大肠杆菌BL21菌株（保藏号H1566）。
上述的重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）应用于催化超氧阴离子自由基，发生歧化反应生成氧气和双氧水。
对本发明的重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）基因所表达的酶分子的氨基酸进行一个或多个氨基酸替换、插入或缺失所得到的蛋白质也能达到本发明的目的。因而本发明还包括与SEQ ID NO: 1-13所示的氨基酸序列具有至少70%的同源性，优选具有至少90%的同源性，但同时具有重组超氧化物歧化酶活性的蛋白质。上面使用的术语“多个”可以是小于100的数目，优选为小于10的数目。
本发明提出的上述重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）的性能不同于已知的超氧化物歧化酶，将这段N段序列添加到其它SOD的N端或sodA功能域的N端，可以提高其它SOD的抗逆性和水溶液中稳定性，可高效催化超氧阴离子自由基(O2·ˉ)发生歧化反应生成氧气O2和双氧水H2O2。
上述的重组超氧化物歧化酶（N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端）改造后的重组超氧化物歧化酶主要应用于医药、卫生保健、食品或化妆品加工。
本发明公开的一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列所具有的积极效果在于：
（1）阐明了一种提高SOD抗逆性和水溶液中稳定性的新方法。利用添加本专利中氨基酸序列的基因重组方法进行SOD的改造和表达，可提高其对高温、极端pH、变性剂、清洁剂、抑制剂等的抗逆性及在水溶液中的稳定性,具有重要应用价值。
（2）通过添加本专利中氨基酸序列所获得的SOD酶具有极高的稳定性，更有利于SOD酶制剂在成分复杂的食品、化妆品、药品和保健品中的添加，并保持长期稳定，延长货架期。
（3）本发明可应用于SOD的工业生产和添加，操作简便、可行性强、适应性好，成本低廉，具有重要的工业应用前景和实际意义。
附图说明：
图1为抑制剂、清洁剂、变性剂对SOD活性的影响。
具体实施方式
下面通过具体实施例并结合附图对本发明作进一步的详细说明。以下各实施例仅仅是用于说明而不是限制本发明。需要特别说明的是实施例中所用到的试剂均由市售，嗜热脱氮芽胞杆菌NG80-2（CGMCC No. 1228）已保藏在国家菌种保藏中心。
实施例1
1．构建编码NG80-2的Fe/Mn-SOD全序列基因（sod-GTNG_2215）的克隆，并且构建编码SOD-GTNG_2215的结构域sodA的DNA序列（sodA-GTNG_2215）的克隆，并且测定抑制剂、清洁剂、变性剂对表达蛋白活性的影响。
1.1 嗜热脱氮芽胞杆菌NG80-2（CGMCC No. 1228）总DNA的提取
在本实施例中，采用从中国天大港油田官69-8区块油井地层水分离获得的嗜热脱氮芽胞杆菌NG80-2（Geobacillus thermodenitrificans该菌株已保藏在中国微生物菌种保藏委员会普通微生物中心，其保藏号为CGMCC No.1228），取其过夜培养的新鲜培养物3mL，离心收集菌体，菌体悬于250μL50mMTris缓冲液中（pH8.0），加入10μL0.4M EDTA（pH8.0），混匀后37℃保温20min，之后加入30μL20mg/L溶菌酶，混匀后37℃再保温20min，再加入5μL20mg/L蛋白酶K，温柔混匀后，再加入20μL10%SDS，50℃保温至溶液澄清，分别用等体积酚:氯仿:异戊醇抽提两次，氯仿:异戊醇抽提一次，最后一次的上清溶液，加入2.5倍体积预冷的无水乙醇，回收DNA，用70%乙醇洗，沉淀溶于100μLTE缓冲液（pH8.0，10mMTris，1 mMEDTA），加入10mg/L RNase 2μL，65℃保温30min，分别用酚:氯仿:异戊醇、氯仿:异戊醇各抽提一次，上清液加入2.5倍体积预冷的无水乙醇，回收DNA，用70%乙醇洗，真空干燥，沉淀溶于50μLTE缓冲液。DNA溶液的紫外分光光度计测定结果为A260/A280=1.95，A260=0.73。
1.2超氧化物歧化酶基因的克隆和筛选
1.2.1扩增NG80-2的Fe/Mn-SOD全序列基因（sod-GTNG_2215），取前面所述的总DNA溶液0.5μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5'GGAATTCCATATGGACGACCAAACGTTGTTTGC 3'
下游引物：5'CCCAAGCTTTTAAAATGGTTGCCAACGCA 3'
1.2.2扩增NG80-2的Fe/Mn-SOD的结构域sodA的DNA序列（sodA-GTNG_2215），取前面所述的总DNA溶液0.5μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5' GGAATTCCATATGCCTGGCAAGCATGTGCTGCC 3'
下游引物：5' CCCAAGCTTTTAAAATGGTTGCCAACGCA 3'
上述两组PCR产物纯化后都用NdeI和HindIII双酶切，分别与经同样限制型内切酶酶解并切胶回收的质粒pET-28a(+)连接，转化感受态大肠杆菌DH5α（本实验室保存）后，涂于含50μg/mL Kan（卡拉霉素）的LB固体培养基上。37℃培养16～18小时，挑取单克隆菌落鉴定，插入有sod-GTNG_2215编码的DNA序列的pET-28a(+)质粒为重组质粒pLW01，含有该质粒的重组大肠杆菌DH5α为DH01。插入有sodA-GTNG_2215编码的序列的pET-28a(+)质粒为重组质粒pLW02，含有该质粒的重组大肠杆菌DH5α为DH02。采用Sanger双脱氧法对此DNA片段进行了测序，测序结果显示插入的DNA序列正确。然后将重组质粒pLW01和pLW02分别转化入大肠杆菌BL21中，此大肠杆菌BL21分别命名为BL01和BL02。
1.3重组超氧化物歧化酶的纯化和特性
将上述重组菌BL01和BL02单克隆分别接入20mL含50μg/mL Kan的LB培养基中，37℃，180rpm/min培养12小时，然后将培养物按1%（V/V）接种量接入200mL含50μg/mL Kan的LB培养基（共2个摇瓶），37℃，220rpm/min培养A600为0.6时，加入IPTG至终浓度为0.1mM, 37℃, 180rpm/min诱导3小时。离心收集菌体，悬于50mMTris-Cl（pH8.0）缓冲液中，利用超声波破碎细胞，离心上清液为重组超氧化物歧化酶的粗提液。此上清液经螯合琼脂糖凝胶（Chelating Sepharose）镍亲合柱层析纯化，得到的酶制剂在SDS-PAGE上显示一条带。理论上推算SOD-GTNG_2215和SODA-GTNG_2215的分子量分别为54.0kD和26.6kD，与SDS-PAGE检测结果一致。
1.4 重组超氧化物歧化酶（SOD）活性测定
3mL反应混合液中14.5mM L-甲硫氨酸加入2.7mL，30μL EDTA-Na2加入10ul，2.25mM NBT加入100uL，60mM核黄素加入100μL，PBS加入90μL，加入10μL的样品酶液。各试剂加完后充分混匀，取1管置于暗处，560nm比色时调零。另取1管不加蛋白酶，用磷酸钠缓冲液代替作为空白对照。其余几管待测样品置于一定温度光强为4000Lux条件下光照15min，然后立刻避光终止反应。在560nm波长处比色时，用置于黑暗处的样品液调零，测定各样品管光吸收并记录结果。在一定测定条件下将NBT光还原反应抑制到对照的50%时的酶量作为一个酶活力单位（U）。
1.5 抑制剂、清洁剂、变性剂对SOD活性的影响
将纯化的超氧化物歧化酶（SOD），分别置于终浓度为1mM或10mM的变性剂（乙二胺四乙酸（EDTA）和β-巯基乙醇（β-ME））、0.1%或1%的清洁剂（十二烷基磺酸钠（SDS））和2.5M的变性剂（尿素和盐酸胍）中，25℃保温30分钟，用上述方法测定超氧化物歧化酶的活性。将不加变性剂、清洁剂、变性剂的反应作为对照，测得的酶活力定义为100%。分别计算不同条件下超氧化物歧化酶的剩余酶活性（采用相对酶活性来表示，即在不同条件下的剩余的酶活性占对照酶活性的百分比）。结果表明（见图1），SOD-GTNG_2215对抑制剂、清洁剂和变性剂的抗性要强于SODA-GTNG_2215。其中，SOD-GTNG_2215表现出对变性剂极强的抗性。在终浓度为2.5M的尿素或盐酸胍中温育后，SOD-GTNG_2215的剩余活性分别为93%和86%，而SODA-GTNG_2215的剩余活性仅为60%和43%。
结论：当SOD-GTNG_2215去除N端序列后，其对抑制剂、清洁剂和变性剂的抗性大幅下降。这说明N端序列对SOD-GTNG_2215的抗逆性具有决定性作用。
2. 构建编码B. subtilis BSn5的Mn-SOD全序列基因（sod-BSn5）的克隆，并且构建编码SOD-BSn5的结构域sodA的DNA序列（sodA-BSn5）的克隆，最后还要构建编码重组SOD（SOD-GTNG_2215的N端序列和B. subtilis BSn5的SODA重组结合成重组SOD）全序列基因（sod-combinant）的克隆。并且测定抑制剂、清洁剂、变性剂对表达蛋白活性的影响。
2.1 B. subtilis BSn5总DNA的提取
在本实施例中，取其过夜培养的新鲜培养物3mL，离心收集菌体，菌体悬于250μL50mM Tris缓冲液中（pH8.0），加入10μL0.4M EDTA（pH8.0），混匀后37℃保温20min，之后加入30μL20mg/L溶菌酶，混匀后37℃再保温20min，再加入5μL20mg/L蛋白酶K，温柔混匀后，再加入20μL10%SDS，50℃保温至溶液澄清，分别用等体积酚:氯仿:异戊醇抽提两次，氯仿:异戊醇抽提一次，最后一次的上清溶液，加入2.5倍体积预冷的无水乙醇，回收DNA，用70%乙醇洗，沉淀溶于100μLTE缓冲液（pH8.0，10mMTris，1mM EDTA），加入10mg/L RNase 2μL，65℃保温30min，分别用酚:氯仿:异戊醇、氯仿:异戊醇各抽提一次，上清液加入2.5倍体积预冷的无水乙醇，回收DNA，用70%乙醇洗，真空干燥，沉淀溶于50μLTE缓冲液。DNA溶液的紫外分光光度计测定结果为A260/A280=1.96，A260=0.72。
2.2超氧化物歧化酶（SOD）基因的克隆和筛选
2.2.1 扩增BSn5的Mn-SOD全序列基因（sod-BSn5），取前面所述的总DNA溶液0.5μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5'GGAATTCATGAAACGTGAATCTTATCAAACG 3'
下游引物：5'CCGCTCGAGTTAATAGAGCTTCCAAACGACTTC 3'
2.2.2扩增BSn5的Mn-SOD的结构域sodA的DNA序列（sodA -BSn5），取前面所述的总DNA溶液0.5μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5' GGAATTCCATATGAAACAcGTGCTGCCAAAGCT 3'
下游引物：5' CGCGGATCCTTAATAGAGCTTCCAAACGACTTC 3'
2.2.3扩增重组SOD的DNA序列（sod-combinant）
2.2.3.1 扩增NG80-2 SOD的N端序列基因（sod_N-GTNG_2215），取前面所述的总DNA溶液0.5μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5'GGAATTCCATATGGACGACCAAACGTTGTTTGC3'
下游引物：5'GCACATGTTTCGAAACCGCC3'
2.2.3.2 扩增BSn5 SOD的C端序列基因（sod_C-BSn5），取前面所述的总DNA溶液0.5μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5'GGCGGTTTCGAAACATGTGC3'
下游引物：5'CGCGGATCCTTAATAGAGTTTCCAAACGACTTC3'
2.2.3.3 扩增重组SOD的DNA序列（sod-combinant），sod_N-GTNG_2215和sod_C-BSn5各取0.25μL（约10ng）作为模板，以下列寡核苷酸序列作为引物，并按下述设定的PCR循环参数进行25个循环PCR。
设定的PCR循环参数如下：
95℃，3min；95℃，30s；55℃，45s；72℃，2min；72℃，10min；4℃，2hr
上游引物：5'GGAATTCCATATG GACGACCAAACGTTGTTTGC 3'
下游引物：5' CGCGGATCCTTAATAGAGcTTCCAAACGACTTC 3'
sod-BSn5的PCR产物纯化后用EcoRI/ XhoI双酶切，sodA-BSn5和sod-combinant的PCR产物纯化后用NdeI/ BamH双酶切，以上酶切产物分别与经同样限制型内切酶酶解并切胶回收的质粒pET-28a(+)连接，转化感受态大肠杆菌DH5α（本实验室保存）后，涂于含50μg/mL Kan（卡拉霉素）的LB固体培养基上。37℃培养16～18小时，挑取单克隆菌落鉴定，插入有sod-BSn5编码的DNA序列的pET-28a(+)质粒为重组质粒pLW03，含有该质粒的重组大肠杆菌DH5α为DH03。插入有sodS-BSn5编码的DNA序列的pET-28a(+)质粒为重组质粒pLW04，含有该质粒的重组大肠杆菌DH5α为DH104。插入有sod-combinant编码的DNA序列的pET-28a(+)质粒为重组质粒pLW05，含有该质粒的重组大肠杆菌DH5α为DH05。采用Sanger双脱氧法对此DNA片段进行了测序，测序结果显示插入的DNA序列正确。将上述重组质粒pLW03、pLW04和 pLW05分别转化入大肠杆菌BL21中，此大肠杆菌BL21分别命名为BL03、BL04和BL05。
2.3 重组超氧化物歧化酶的纯化和特性
将上述重组菌BL03、BL04和BL05单克隆分别接入20mL含50μg/mL Kan的LB培养基中，37℃，180rpm/min培养12小时，然后将培养物按1%（V/V）接种量接入200mL含50μg/mL Kan的LB培养基（共2个摇瓶），37℃，220rpm/min培养A600为0.6时，BL03加入IPTG至终浓度为0.05mM, 25℃, 180rpm/min诱导3小时。BL04加入IPTG至终浓度为0.05mM, 25℃, 180rpm/min诱导3小时。BL05加入IPTG至终浓度为0.1mM, 30℃, 180rpm/min诱导3小时。诱导完后离心收集菌体，悬于50mMTris-Cl（pH8.0）缓冲液中，利用超声波破碎细胞，离心上清液为重组超氧化物歧化酶的粗提液。此上清液经螯合琼脂糖凝胶（Chelating Sepharose）镍亲合柱层析纯化，得到的酶制剂在SDS-PAGE上显示一条带。理论上推算SOD-BSn5、SODA-BSn5 和 SOD-combinant的分子量分别为37.28099 kD、26.28290 kD和53.61853 kD，与SDS-PAGE检测结果一致。
2.4 重组超氧化物歧化酶活性测定
3mL反应混合液中14.5 mML-甲硫氨酸加入2.7mL，30μL EDTA-Na2加入10μL，2.25 mM NBT加入100μL，60μM核黄素加入100μL，PBS加入90μL，加入10μL的样品酶液。各试剂加完后充分混匀，取1管置于暗处，560nm比色时调零。另取1管不加蛋白酶，用磷酸钠缓冲液代替作为空白对照。其余几管待测样品置于一定温度光强为4000Lux条件下光照15min，然后立刻避光终止反应。在560nm波长处比色时，用置于黑暗处的样品液调零，测定各样品管光吸收并记录结果。在一定测定条件下将NBT光还原反应抑制到对照的50%时的酶量作为一个酶活力单位（U）
2.5 抑制剂、清洁剂、变性剂对SOD活性的影响
将纯化的超氧化物歧化酶（SOD），分别置于终浓度为1mM或10mM的变性剂（乙二胺四乙酸（EDTA）和β-巯基乙醇（β-ME））、0.1%或1%的清洁剂（十二烷基磺酸钠（SDS））和2.5M的变性剂（尿素和盐酸胍）中，25℃保温30分钟，用上述方法测定超氧化物歧化酶的活性。将不加变性剂、清洁剂、变性剂的反应作为对照，测得的酶活力定义为100%。分别计算不同条件下超氧化物歧化酶的剩余酶活性（采用相对酶活性来表示，即在不同条件下的剩余的酶活性占对照酶活性的百分比）。结果表明（见图1），SOD-combinant对抑制剂、清洁剂和变性剂的抗性要强于SOD-BSn5和SODA-BSn5。其中，SOD-combinant表现出对变性剂极强的抗性。在终浓度为2.5M的尿素或盐酸胍中温育后，SOD-combinant的剩余活性分别为71%和90%，而SOD-BSn5、SODA-BSn5的剩余活性仅为44%~64%。
结论：N端氨基酸序列添加到其它SOD（SODA-BSn5）的N端，可以显著提高其SOD对抑制剂、清洁剂和变性剂的抗性。
实施例2
构建编码B. subtilis BSn5的Mn-SOD全序列基因（sod-BSn5）的克隆，并且构建编码重组SOD（SOD-GTNG_2215的N端序列和B. subtilisBSn5的SODA重组结合成重组SOD）全序列基因（sod-combinant）的克隆。并测定酶在水溶液中及制成冻干粉常温存放的稳定性。
1. 超氧化物歧化酶（SOD）基因的克隆构建、蛋白纯化及酶活性测定
具体方法见实施例1的2。
2. 超氧化物歧化酶在水溶液中稳定性
取浓度为50mg/ml的酶液10ml，常温（25℃）存放于密闭容器内。分别在不同的时间（15天，30天，180天，360天），测定超氧化物歧化酶的活性，并观察酶蛋白析出情况。将0天的酶活力定义为100%，分别计算各个时间点超氧化物歧化酶的剩余酶活性（采用相对酶活性来表示，即在不同时间点的剩余的酶活性占0天酶活性的百分比）。结果表明（见表1），sod-BSn5半衰期大于15天，且保存15天时有酶蛋白析出，180天时析出沉淀量增多；sod-combinant半衰期大于360天，且保存360天时酶溶液仍稳定，无沉淀析出：
表1 SOD在水溶液中的稳定性：
注:＋为沉淀;－为无沉淀生成，NA表示未检测到活性
3. 常温存放酶活
将上述得到的SOD进行冷冻干燥，在常温（25℃）存放360天，检测SOD的剩余酶活性（将0天的酶活力定义为100%，采用相对酶活性来表示，即存放360天的剩余的酶活性占0天酶活性的百分比）。结果表明（见表2），存放360天，sod-BSn5活力损耗小于10%，sod-combinant活力损耗小于5：
结论：N端氨基酸序列添加到其它SOD（SODA-BSn5）的N端，可显著提高蛋白酶在水溶液中的稳定性，延长冻干粉存放周期。
<110> 南开大学
<120>一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列及其应用
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<170> PatentIn version 3.5
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<213> Geobacillus thermodenitrificans NG80-2
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<211> 344
<212> PRT
<213> Geobacillus thermoleovorans CCB_US3_UF5
<400> 2
Met Arg Leu Asp Lys Glu Thr Val Trp Cys Ser Arg Arg Phe Ala Lys
1 5 10 15
Ala Ala Cys Leu Phe Gln Lys Lys Gly Gly Ala Asp Pro Lys Arg Glu
20 25 30
Tyr Val Leu Gly Gln Pro Asp Phe Phe Glu Arg Phe Ile Tyr Gly Ser
35 40 45
Phe Ala Asp Pro Leu Gln Gly Ser Ile Thr Ala Ile Leu Phe Phe Leu
50 55 60
Ala Val Phe Ser Tyr Asp Asp Cys Pro Arg Ser Ser Met Tyr Glu Arg
65 70 75 80
Pro Gly Ile Trp Val Asn Ala Asp Lys Asn Asp Phe Pro Met Thr Ala
85 90 95
Tyr Asp Met Ala Asn Lys Thr Gly Gly Gly Phe Tyr Gln Met Asp Asp
100 105 110
Gln Val Leu Phe Ala Gln Tyr Ala Ala Arg Val Asn Glu Trp Gly Asn
115 120 125
Gln Val Lys Glu Thr Leu Ala Leu Arg Gly Ala Ser Thr Asp Gly Ala
130 135 140
Ser Ser Leu Leu Glu Phe Ile Ala Glu His Asp Gly Glu Trp Thr Glu
145 150 155 160
Glu Ala Val Arg Glu Leu Gln Arg Leu Ala Asp Asp Val Tyr Val Gly
165 170 175
Ala Leu Arg Gln Tyr Val Met Glu Ala Ala Ala Trp Gly Arg Gln Val
180 185 190
Glu Gln Ala Leu Ser Ala Arg Arg Met Ala Glu Asp Val Gly Leu Ser
195 200 205
Ser Leu Leu Ala Tyr Ile Asp Gly His Gly Asp Glu Trp Thr Glu Glu
210 215 220
Ala Ile Tyr Glu Leu Gln Arg Leu Val Asp Asp Val Tyr Thr Arg Ala
225 230 235 240
Val Arg Leu Ala Asp Ser Ser Ala Ala Asp Arg Glu Glu Glu Ala Thr
245 250 255
Gln Glu Gln Val Glu Gly Glu Ser Val Ser Pro Glu Leu Glu Ser Glu
260 265 270
Asn Lys Glu Asn Glu Asp Gly Trp Leu Asp Thr Ser Gly Thr Ala Glu
275 280 285
Arg Val Glu Asp Ala Lys Glu Pro Ala Phe Met Ala Glu Leu Ser Asp
290 295 300
Ser Pro Thr Asp Ala Ala Asp Gly Glu Pro Asp Gln Ala Asp Asn Val
305 310 315 320
Thr Asp Gly Lys Arg Arg Trp Val Asp Ala Asp Asp Gly Gly Glu Pro
325 330 335
Arg Gln Gln Val Ala Pro Gly Arg
340
<210> 3
<211> 244
<212> PRT
<213> Geobacillus sp. G11MC16
<400> 3
Met Asp Asp Gln Ala Leu Phe Ala Gln Tyr Ala Ala Glu Val Asn Glu
1 5 10 15
Trp Gly Glu Gln Val Lys Gln Val Leu Glu Leu Arg Gly Ala Ser Ile
20 25 30
Asp Gly Ala Ser Thr Leu Leu Gln Phe Ile Ala Glu His Asp Gly Glu
35 40 45
Trp Thr Glu Glu Ala Val Arg Glu Leu Thr Arg Leu Val Asp Asp Val
50 55 60
Tyr Ala Ala Ala Leu Arg His Tyr Ala Ile Glu Ala Ala Glu Trp Gly
65 70 75 80
Lys Gln Val Glu His Ala Leu Ser Met Arg Gly Ala Ala Glu Asp Ile
85 90 95
Gly Leu Ser Ser Leu Leu Ala Arg Ile Glu Glu His Gly Asp Glu Trp
100 105 110
Thr Glu Glu Glu Ile His Glu Leu Gln Leu Leu Val Asp Asp Val Tyr
115 120 125
Ala Arg Ala Ile Arg Leu Val Glu Pro Leu Ser Asp Gly Gln Glu Glu
130 135 140
Asp Leu Thr Arg Gln Glu Glu Val Ser Ala Leu Pro Glu Gln Glu Gly
145 150 155 160
Gly Asn Gly Glu Gln Met Ser Glu Gly Thr Glu Arg Ser Gly Glu His
165 170 175
Lys Gly Asp Ser Glu Gln Glu Pro Val Val Ala Ala Glu Arg Ala Glu
180 185 190
Pro Phe Ile Ala Ser Ser Thr Asp Ser Pro Asp Gly Glu Gln Leu His
195 200 205
Glu Gly Asp Thr Met Asp Glu Glu Trp Arg His Asn Ala Asp Met Thr
210 215 220
Asp Lys Glu Arg Leu Thr Glu Glu Gly Val Thr Asp Gly Glu Arg Gln
225 230 235 240
Arg Ala Val Ser
<210> 4
<211> 235
<212> PRT
<213> Geobacillus sp. C56-T3
<400> 4
Met Asp Asp Gln Val Leu Phe Ala Gln Tyr Ala Ala Arg Val Asn Glu
1 5 10 15
Trp Gly Asn Gln Val Lys Glu Thr Leu Ala Leu Arg Gly Ala Ser Thr
20 25 30
Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile Ala Glu His Asp Gly Glu
35 40 45
Trp Thr Glu Glu Ala Val Arg Glu Leu Gln Arg Leu Ala Asp Asp Val
50 55 60
Tyr Val Gly Ala Leu Arg Gln Tyr Val Met Glu Ala Ala Ala Trp Gly
65 70 75 80
Arg Gln Val Glu Gln Ala Leu Ser Ala Arg Arg Met Ala Glu Asp Val
85 90 95
Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp Gly His Gly Asp Glu Trp
100 105 110
Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg Leu Val Asp Asp Val Tyr
115 120 125
Thr Arg Ala Val Arg Leu Ala Asp Ser Ser Ala Ala Glu Arg Glu Glu
130 135 140
Glu Ala Thr Gln Glu Gln Val Glu Gly Glu Ser Val Ser Pro Glu Leu
145 150 155 160
Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly Trp Leu Asp Thr Ser Gly
165 170 175
Thr Ala Glu Arg Val Glu Asp Ala Lys Glu Pro Ala Phe Met Ala Glu
180 185 190
Leu Ser Asp Ser Leu Pro Asp Ile Ala Asp Gly Glu Pro Gly Gln Ala
195 200 205
Asp Asn Val Thr Asp Gly Lys Arg Arg Trp Val Asp Ala Asp Asp Gly
210 215 220
Gly Glu Pro Arg Gln Gln Ala Ala Pro Gly Arg
225 230 235
<210> 5
<211> 235
<212> PRT
<213> Geobacillus sp. Y412MC52
<400> 5
Met Asp Asp Gln Val Leu Phe Ala Gln Tyr Ala Ala Arg Val Asn Glu
1 5 10 15
Trp Gly Asn Gln Val Lys Glu Thr Leu Ala Leu Arg Gly Ala Ser Thr
20 25 30
Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile Ala Glu His Asp Gly Glu
35 40 45
Trp Thr Glu Glu Ala Val Arg Glu Leu Gln Arg Leu Ala Asp Asp Val
50 55 60
Tyr Val Gly Ala Leu Arg Gln Tyr Val Met Glu Ala Ala Ala Trp Gly
65 70 75 80
Arg Gln Val Glu Gln Ala Leu Ser Ala Arg Arg Met Ala Glu Asp Val
85 90 95
Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp Gly His Gly Asp Glu Trp
100 105 110
Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg Leu Val Asp Asp Val Tyr
115 120 125
Thr Arg Ala Val Arg Leu Ala Asp Ser Ser Ala Ala Glu Arg Glu Glu
130 135 140
Glu Ala Thr Gln Glu Gln Val Glu Gly Glu Ser Val Ser Pro Glu Leu
145 150 155 160
Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly Trp Leu Asp Thr Ser Gly
165 170 175
Thr Ala Glu Arg Val Glu Asp Ala Lys Glu Pro Ala Phe Met Ala Glu
180 185 190
Leu Ser Asp Ser Leu Pro Asp Ile Ala Asp Gly Glu Pro Gly Gln Ala
195 200 205
Asp Asn Val Thr Asp Gly Lys Arg Arg Trp Val Asp Ala Asp Asp Gly
210 215 220
Gly Glu Pro Arg Gln Gln Val Ala Pro Gly Arg
225 230 235
<210> 6
<211> 235
<212> PRT
<213> Geobacillus sp. GHH01
<400> 6
Met Asp Asp Gln Val Leu Phe Ala Gln Tyr Ala Ala Arg Val Asn Glu
1 5 10 15
Trp Gly Asn Gln Val Lys Glu Thr Leu Ala Leu Arg Gly Ala Ser Thr
20 25 30
Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile Ala Glu His Asp Gly Glu
35 40 45
Trp Thr Glu Glu Ala Val Arg Glu Leu Gln Arg Leu Ala Asp Asp Val
50 55 60
Tyr Val Gly Ala Leu Arg Gln Tyr Val Met Glu Ala Ala Ala Trp Gly
65 70 75 80
Arg Gln Val Glu Gln Ala Leu Ser Ala Arg Arg Met Ala Glu Asp Val
85 90 95
Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp Gly His Gly Asp Glu Trp
100 105 110
Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg Leu Val Asp Asp Val Tyr
115 120 125
Thr Arg Ala Val Arg Leu Ala Asp Ser Ser Ala Ala Glu Arg Glu Glu
130 135 140
Glu Ala Thr Gln Glu Gln Val Glu Gly Glu Ser Val Ser Pro Glu Leu
145 150 155 160
Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly Trp Leu Asp Thr Ser Gly
165 170 175
Thr Ala Glu Arg Val Glu Asp Ala Lys Glu Pro Ala Phe Met Ala Glu
180 185 190
Leu Ser Asp Ser Leu Pro Asp Ile Ala Asp Gly Glu Pro Gly Gln Ala
195 200 205
Asp Asn Val Thr Asp Gly Lys Arg Arg Trp Val Asp Ala Asp Asp Gly
210 215 220
Gly Glu Pro Arg Gln Gln Ala Ala Pro Gly Arg
225 230 235
<210> 7
<211> 235
<212> PRT
<213> Geobacillus sp. Y412MC61
<400> 7
Met Asp Asp Gln Val Leu Phe Ala Gln Tyr Ala Ala Arg Val Asn Glu
1 5 10 15
Trp Gly Asn Gln Val Lys Glu Thr Leu Ala Leu Arg Gly Ala Ser Thr
20 25 30
Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile Ala Glu His Asp Gly Glu
35 40 45
Trp Thr Glu Glu Ala Val Arg Glu Leu Gln Arg Leu Ala Asp Asp Val
50 55 60
Tyr Val Gly Ala Leu Arg Gln Tyr Val Met Glu Ala Ala Ala Trp Gly
65 70 75 80
Arg Gln Val Glu Gln Ala Leu Ser Ala Arg Arg Met Ala Glu Asp Val
85 90 95
Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp Gly His Gly Asp Glu Trp
100 105 110
Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg Leu Val Asp Asp Val Tyr
115 120 125
Thr Arg Ala Val Arg Leu Ala Asp Ser Ser Ala Ala Glu Arg Glu Glu
130 135 140
Glu Ala Thr Gln Glu Gln Val Glu Gly Glu Ser Val Ser Pro Glu Leu
145 150 155 160
Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly Trp Leu Asp Thr Ser Gly
165 170 175
Thr Ala Glu Arg Val Glu Asp Ala Lys Glu Pro Ala Phe Met Ala Glu
180 185 190
Leu Ser Asp Ser Leu Pro Asp Ile Ala Asp Gly Glu Pro Gly Gln Ala
195 200 205
Asp Asn Val Thr Asp Gly Lys Arg Arg Trp Val Asp Ala Asp Asp Gly
210 215 220
Gly Glu Pro Arg Gln Gln Val Ala Pro Gly Arg
225 230 235
<210> 8
<211> 209
<212> PRT
<213> Geobacillus kaustophilus HTA426
<400> 8
Met Arg Gly Ala Ser Thr Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile
1 5 10 15
Ala Glu His Asp Gly Glu Trp Thr Glu Glu Ala Val Arg Glu Leu Gln
20 25 30
Arg Leu Ala Asp Asp Val Tyr Val Gly Ala Leu Arg Gln Tyr Val Met
35 40 45
Glu Ala Ala Ala Trp Gly Arg Gln Val Glu Gln Ala Leu Ser Ala Arg
50 55 60
Arg Met Ala Glu Asp Val Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp
65 70 75 80
Gly His Gly Asp Glu Trp Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg
85 90 95
Leu Val Asp Asp Val Tyr Thr Arg Ala Val Arg Leu Ala Asp Ser Ser
100 105 110
Ala Ala Asp Arg Glu Glu Glu Ala Thr Gln Glu Gln Val Glu Gly Glu
115 120 125
Ser Val Ser Pro Glu Leu Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly
130 135 140
Trp Leu Asp Thr Ser Gly Thr Ala Glu Arg Val Glu Asp Ala Lys Glu
145 150 155 160
Pro Ala Phe Met Ala Glu Leu Ser Asp Ser Pro Thr Asp Ala Ala Asp
165 170 175
Gly Glu Pro Asp Gln Ala Asp Asn Val Thr Asp Gly Lys Arg Arg Trp
180 185 190
Val Asp Ala Asp Asp Gly Gly Glu Pro Arg Gln Gln Val Ala Pro Gly
195 200 205
Arg
<210> 9
<211> 209
<212> PRT
<213> Geobacillus sp. POT5
<400> 9
Leu Arg Gly Ala Ser Thr Asp Gly Ala Ser Ser Leu Leu Glu Phe Ile
1 5 10 15
Ala Glu His Asp Gly Glu Trp Thr Glu Glu Ala Val Arg Glu Leu Gln
20 25 30
Arg Leu Ala Asp Asp Val Tyr Val Gly Ala Leu Arg Gln Tyr Val Met
35 40 45
Glu Ala Ala Ala Trp Gly Arg Gln Val Glu Gln Ala Leu Ser Ala Arg
50 55 60
Arg Met Ala Glu Asp Val Gly Leu Ser Ser Leu Leu Ala Tyr Ile Asp
65 70 75 80
Gly His Gly Asp Glu Trp Thr Glu Glu Ala Ile Tyr Glu Leu Gln Arg
85 90 95
Leu Val Asp Asp Val Tyr Thr Arg Ala Val Arg Leu Ala Asp Ser Ser
100 105 110
Ala Ala Asp Arg Glu Glu Gly Ala Thr Gln Glu Gln Val Glu Gly Glu
115 120 125
Ser Val Ser Pro Glu Leu Glu Ser Glu Asn Lys Glu Asn Glu Asp Gly
130 135 140
Trp Leu Asp Thr Ser Gly Thr Ala Glu Arg Val Glu Asp Ala Lys Glu
145 150 155 160
Pro Ala Phe Met Ala Glu Leu Ser Asp Ser Pro Thr Asp Ala Ala Asp
165 170 175
Gly Glu Pro Asp Gln Val Asp Asn Val Thr Asp Gly Lys Arg Arg Trp
180 185 190
Val Asp Ala Asp Asp Gly Gly Glu Pro Arg Gln Gln Val Ala Pro Gly
195 200 205
Arg
<210> 10
<211> 146
<212> PRT
<213> Geobacillus sp. WCH70
<400> 10
Met Asn Glu Gln Glu Arg Ile Gln Gln Tyr Val Ala Glu Val Lys Glu
1 5 10 15
Trp Gly Lys Gln Val Glu Gln Ile Leu Leu Gln Arg Gly Glu Asp Gly
20 25 30
Gly Asp Cys Arg Val Asp Ser Leu Leu Ser Tyr Ile Glu His His Gly
35 40 45
Asp Ala Trp Thr Glu Asp Ala Ile Tyr Glu Leu Gln Arg Met Val Asp
50 55 60
Glu Val Tyr Glu Lys Ala Leu Val Phe Gln Gln Asn Gly Gln Ser Ala
65 70 75 80
Ile Arg Gln Glu Glu Ser Gly Thr Glu Glu Arg Gln Gln Thr Ser Ile
85 90 95
Gly Gln Glu Glu Asn Gly Ile Glu Glu Arg Gln Gln Thr Ala Val Arg
100 105 110
Gln Glu Glu Ser Gly Thr Glu Glu Arg Gln Gln Thr Ala Ser Glu Gln
115 120 125
Glu Glu Glu Ser Glu Ala Glu Glu Arg Gln Gln Thr Tyr Val Ala Ala
130 135 140
Gly Arg
145
<210> 11
<211> 101
<212> PRT
<213> Geobacillus thermoglucosidans TNO-09.020
<400> 11
Met Ser Glu Gln Glu Leu Phe Gln Arg Tyr Val Lys Gln Val Ser Glu
1 5 10 15
Trp Gly Ala Gln Val Gly Gln Met Leu Pro Arg Arg Asp Asp Gly Thr
20 25 30
Val His His Asp Ile Ala Ala Leu Leu Ser Tyr Ile Asp Arg His Asp
35 40 45
Gly Glu Trp Thr Glu Thr Glu Ile Tyr Asp Leu Gln Arg Met Ala Asp
50 55 60
Ala Val Tyr Glu Lys Ala Ala Ala Val Ser Ala Asn Gly Thr Leu Ala
65 70 75 80
Asp Glu Ser Arg Glu Thr Ser Glu Glu Glu Ala Arg Arg Gln Thr Tyr
85 90 95
Ile Thr Ala Gly Arg
100
<210> 12
<211> 101
<212> PRT
<213> Geobacillus thermoglucosidasius C56-YS93
<400> 12
Met Ser Glu Gln Glu Leu Phe Gln Arg Tyr Val Lys Gln Val Ser Glu
1 5 10 15
Trp Gly Ala Gln Val Gly Gln Met Leu Pro Arg Arg Asp Asp Gly Thr
20 25 30
Val His His Asp Ile Ala Ala Leu Leu Ser Tyr Ile Asp Arg His Asp
35 40 45
Gly Glu Trp Thr Glu Thr Glu Ile Tyr Asp Leu Gln Arg Met Ala Asp
50 55 60
Ala Val Tyr Glu Lys Ala Ala Ala Val Ser Ala Asn Gly Thr Leu Ala
65 70 75 80
Asp Glu Ser Arg Glu Thr Ser Glu Glu Glu Ala Arg Arg Gln Thr Tyr
85 90 95
Ile Thr Ala Gly Arg
100
<210> 13
<211> 101
<212> PRT
<213> Geobacillus sp. Y4.1MC1
<400> 13
Met Ser Glu Gln Glu Leu Phe Gln Arg Tyr Val Lys Gln Val Ser Lys
1 5 10 15
Trp Gly Ala Gln Val Gly Gln Met Leu Pro Arg Arg Asp Asp Gly Thr
20 25 30
Val His Arg Asp Ile Ala Ala Leu Leu Ser Tyr Ile Asp Arg His Asp
35 40 45
Gly Glu Trp Thr Glu Thr Glu Ile Tyr Asp Leu Gln Arg Met Ala Asp
50 55 60
Ala Val Tyr Glu Lys Ala Ala Ala Val Ser Ala Asn Gly Thr Leu Ala
65 70 75 80
Asp Glu Ser Arg Glu Thr Ser Glu Glu Glu Ala Arg Arg Gln Thr Tyr
85 90 95
Ile Thr Ala Gly Arg
100
Claims (6)
1.一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列，其特征在于它是来源于Geobacillus属的13个特殊嗜热Fe/Mn-SOD的N端氨基酸序列，如SEQ ID NO:1-13所示。
2.权利要求1所述的一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列，其特征在于：
（1）这组N端氨基酸序列来源于Geobacillus属的13个菌株，其中，SEQ ID NO:1来源于Geobacillus thermodenitrificans NG80-2；SEQ ID NO:2来源于Geobacillus thermoleovorans CCB_US3_UF5；SEQ ID NO:3来源于Geobacillus sp. G11MC16； SEQ ID NO:4来源于Geobacillus sp. C56-T3；SEQ ID NO:5来源于Geobacillus sp. Y412MC52；SEQ ID NO:6来源于Geobacillus sp. GHH01；SEQ ID NO:7来源于Geobacillus sp. Y412MC61；SEQ ID NO:8来源于Geobacillus kaustophilus HTA426；SEQ ID NO:9来源于Geobacillus sp. POT5；SEQ ID NO:10来源于Geobacillus sp. WCH70；SEQ ID NO:11来源于Geobacillus thermoglucosidansTNO-09.020；SEQ ID NO:12来源于Geobacillus thermoglucosidasius C56-YS93；SEQ ID NO:13来源于Geobacillus sp. Y4.1MC1；
（2）这组N端氨基酸序列位于Fe/Mn-SOD蛋白的sodA功能域之前，序列长度分别为244个氨基酸（SEQ ID NO: 1）、344个氨基酸（SEQ ID NO:2）、244个氨基酸（SEQ ID NO:3）、235个氨基酸（SEQ ID NO:4-7）、209个氨基酸（SEQ ID NO:8-9）、146个氨基酸（SEQ ID NO:10）、101个氨基酸（SEQ ID NO:11-13）；
（3）这组N端氨基酸序列相互之间具有85-100%的同源性；
（4）这组N端氨基酸序列都包含特殊的共有重复序列Geo-N-repeat，其中SEQ ID NO:1-9的9个N端序列都含有2个重复序列，SEQ ID NO:10-13的4个N端序列都含有1个重复序列。
3.权利要求1所述的一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列，其中所述N端多肽序列具有与SEQ ID NO:1-13所示的氨基酸序列至少70%的同源性；SEQ ID NO: 1-13所示的氨基酸序列中缺失、替换或插入一个或多个氨基酸后的氨基酸序列，并且具有该序列的蛋白质有对Geobacillus属的Fe/Mn-SOD的抗逆性和水溶液中稳定性具有决定性作用。
4.根据权利要求1至3中任一项所述的一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列，其特征在于，这组N端氨基酸序列可广泛应用于其它SOD，特别是传统工业用SOD的抗逆性和水溶液中稳定性改造；通过一定方法，将这段N端氨基酸序列添加到其它SOD的N端或sodA功能域的N端。
5.权利要求1至3中任一项所述的一组可提高SOD抗逆性和水溶液中稳定性的氨基酸序列，其特征在于将编码N端氨基酸序列的核苷酸与编码其它SOD或sodA功能域的核苷酸连接后经过克隆表达重组SOD，可显著提高SOD的抗逆性和水溶液中稳定性。
6.权利要求1所述的SEQ ID NO:1-13氨基酸序列在提高SOD的抗逆性、提高SOD在水溶液中方面的应用。
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