CN104099352B - 一种高活性细菌硝基还原酶基因及其应用 - Google Patents
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Abstract
本发明公开了一种突变型细菌硝基还原酶基因及其应用,属于酶工程领域。根据多序列比对及蛋白结构分析,设计突变位点,以来自大肠杆菌的硝基还原酶NfsB基因为模板,采用重叠延伸PCR技术将NfsB基因第123位的苯丙氨酸(Phe)突变为丙氨酸(Ala)。突变后123位苯丙氨酸的空间位阻效应被解除,更有利于硝基类底物的结合和催化。本发明所述硝基还原酶突变体F123A具有硝基还原酶活性,与野生型硝基还原酶相比催化效率显著提高2-5倍。本发明所提供硝基还原酶突变体可广泛应用于药物激活及代谢、芳香羟胺合成及硝基污染物降解等诸多领域。
Description
技术领域
本发明属于酶工程领域,具体涉及一种突变型硝基还原酶的基因,以及含有该基因的重组表达转化体作为催化剂的应用。
背景技术
硝基还原酶是一类依赖于FMN或FAD的细胞质酶,可利用NAD(P)H实现硝基芳香族化合物和硝基杂环衍生物的还原代谢。该酶能够催化多种底物的硝基还原反应,在药物激活、羟氨合成及污染物生物代谢等诸多领域有着广泛的应用。AnlezarkGM等人发现,大肠杆菌硝基还原酶NfsB能够激活癌症前药5-(1-氮丙啶)-2,4-二硝基苯甲酰胺(CB1954),使其转化成为强细胞毒性的DNA交联剂,可同时杀死增殖期和休眠期的癌细胞。此外,硝基还原酶可在常温常压的条件下通过对苯环上硝基的还原,生成重要的羟胺类药物中间体或实现污染物的有效降解。相对于传统的物理和化学处理法外,硝基还原酶催化法反应条件温和、成本低且环境友好。
尽管硝基还原酶在催化反应中具有明显的优势,但催化效率低成为制约其广泛应用的主要因素。如NfsB-CB1954前药激活系统虽已进入三期临床阶段,但体内激活效率很低。CB1954对于NfsB而言是一个相当差的底物,Km值高达860μM。临床实验中当药剂量达到人体承受最高值时,病人体内可达到的CB1954血药峰度仅为5-10μM,比Km低大约100倍[5]。因此,如果能降低NfsB对CB1954的Km值,提高催化效率,将对NfsB-CB1954前药激活系统在临床上的广泛应用具有重要意义。
目前硝基还原酶改造的核心问题是生理底物和催化机制尚不明确,从而极大地限制了对该酶的理性改造。为获得更好催化活性的硝基还原酶,人们通常对所有可能的氨基酸位点采用随机突变的方法构建相应的突变体库,随后对突变库中大量的单点突变体、多点突变体进行活性筛选测定,获得所需要的突变体。虽然利用该方法人们也获得了一些活性较好的突变体,但是缺乏对氨基酸的功能性分析,加之基因突变方向的盲目性和不确定性,使得该过程需要大量的资金和人力。若能利用生物信息学和分子对接等模拟手段对氨基酸位点替换对底物结合和催化活性可能的影响做出预测,再结合体外定点突变实验,将显著提高突变实验的成功率。因此,基于蛋白氨基酸序列及晶体结构信息,利用定点突变手段对硝基还原酶进行理性改造,以获得高催化活性、特异性及热稳定性的应用型硝基还原酶,具有显著的商业价值和应用前景。
发明内容
为改善硝基还原酶催化活性差的现状,本发明基于现有技术中的硝基还原酶的氨基酸序列信息和蛋白晶体结构信息,利用生物信息学和分子对接等模拟手段对该分子中可能与底物结合和催化的关键氨基酸进行保守性和功能性分析,结合定点突变手段对硝基还原酶进行分子改造,以期得到高催化活性的硝基还原酶。具体为通过123位苯丙氨酸的缺失突变对硝基还原酶进行改造,从而为Phe124结合底物时发生位移提供更大的空间,提高酶的催化活性。
目前硝基还原酶的生理底物和催化机制尚不明确。对于外源的硝基芳香化合物而言,无法确定该底物在酶活性口袋内的正确定位方式,酶与底物之间如何进行相互作用以及酶如何实现底物的整个催化反应等问题尚不清楚,从而极大地限制了对硝基还原酶的理性改造。GroveJI等人曾对大肠杆菌硝基还原酶NfsB活性口袋内所有可能的位点进行了随机突变和组合突变筛选,发现仅有少量突变体表现出较好的活性,大多数突变体活性较野生型相比有显著的下降,甚至造成酶活性的丧失。此外,研究发现这些活性较好的突变体虽对测试底物CB1954表现出活性提高的特性,但对其他硝基类底物的催化活性并未得到改善,甚至还有不同程度的降低。
对上述现象分析发现,虽然酶活性口袋内的氨基酸可能直接影响酶对底物结合和催化过程,但由于其保守性较高且通常具有某些重要的生物学功能,对这些位点的突变具有很高的风险性和不确定性。针对这一问题,本发明在对关键氨基酸位点预测的基础上,进一步分析了这些位点的保守性和功能性(图1是NfsB与其他家族成员基因的多序列比对分析),并首次将突变的位点着眼于保守性较低且对底物结合和催化过程起到间接性作用的氨基酸位点。即Phe123位点并不与底物直接发生相互作用,而是通过影响活性口袋内部氨基酸Phe124的作用而间接影响该酶的催化过程。
本发明的一方面在于提供一种突变型硝基还原酶的基因,所述硝基还原酶突变体是123位氨基酸苯丙氨酸Phe突变为丙氨酸Ala,命名为F123A。其核苷酸序列如SEQIDNO:1所示。
本发明的另一目的是提供上述突变型硝基还原酶基因编码的蛋白,氨基酸序列如SEQIDNO:2所示。
本发明的另一目的是提供含有该突变型硝基还原酶基因的重组表达载体及其重组表达转化体。以及编码所述硝基还原酶突变体的DNA序列的基因工程菌或转基因细胞系。
本发明的另一目的是提供一种重组硝基还原酶的制备方法,其步骤包括:培养上述含有该突变型硝基还原酶基因的重组表达转化体,从培养物中纯化得到突变型细菌硝基还原酶的蛋白。
上文所述的制备方法,其更具体的技术方案如下:根据大肠杆菌(Escherichiacoli)硝基还原酶NfsB基因NCBI编码:NC_000913,设计定点突变的突变引物,以携带硝基还原酶基因的克隆载体为模板进行定点突变构建突变体;以pET-28a或能表达该酶的载体为表达载体,将重组质粒转化到大肠杆菌BL21(DE3)细胞或能表达该酶的宿主细胞,挑选验证后的阳性单克隆进行发酵培养。经融合表达纯化得到突变型硝基还原酶蛋白。
本发明的另一目的是提供上述技术方案中所述的突变型硝基还原酶基因编码的蛋白或重组表达转化体,二者作为催化剂在催化硝基还原,在药物激活剂代谢、芳香羟胺化合物的生物合成以及硝基类环境污染物生物代谢等领域的应用。本发明所述的突变体硝基还原催化活性较野生型蛋白显著提高。
附图说明
图1.NfsB与其他家族成员基因的多序列比对分析。由图中所示内容可知,该硝基还原酶家族存在一个高度不保守区域,主要差别存在于α螺旋5和α螺旋6中,而该螺旋构成活性口袋入口处一部分。尤其是其中NfsB上存在两个连续的苯丙氨酸(Phe123和Phe124)。Phe124在家族中保守性较高,但Phe123则表现出明显的差异。连续两个苯丙氨酸的存在可能影响酶对底物的结合及催化。
图2.SDS-PAGE电泳分析重组F123A表达、纯化及westernblotting验证。其中1:IPTG未诱导的细胞破碎液;2:0.5mMIPTG诱导的细胞破碎液;3:Ni柱亲和层析的上样透过液;4:20mM咪唑洗脱液;5:75mM咪唑的洗脱液;6:250mM咪唑的洗脱液。由图中所示内容可知,IPTG诱导后细胞破碎液中出现一条明显的诱导条带,分子量大小约27KDa,与理论分子量大小一致。该蛋白经Ni柱亲和层析纯化,于250mM咪唑洗脱组分中得到纯化富集。同时样品经westernblotting验证该蛋白为重组目的蛋白。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
本发明中设计使用的重叠延伸PCR引物序列:
nfsB-WT-F:5’-GGAATTCCATATGGATATCATTTCTGTCGCCTTAAAG-3’;(SEQIDNO:3)
nfsB-WT-R:5’-GGAATTCTTACACTTCGGTTAAGGTGATGTT-3’;(SEQIDNO:4)
nfsB-F123A-F:5’-CGCAAGGCCTTCGCTGATATGCACCGTAAAGATC-3’;(SEQIDNO:5)
nfsB-F123A-R:5’-ATATCAGCGAAGGCCTTGCGACCTTTATCGTTCG-3’(SEQIDNO:6)
实施例1突变型细菌还原酶F123A基因获取及表达载体构建
1.野生型nfsB基因克隆与克隆载体构建
根据NCBI提供的E.coliK12菌株(Invitrogen)中nfsB基因序列,设计引物(nfsB-WT-F:5’-GGAATTCCATATGGATATCATTTCTGTCGCCTTAAAG-3’,nfsB-WT-R:5’-GGAATTCTTACACTTCGGTTAAGGTGATGTT-3’),其中下划线部分表示酶切位点对应的基因序列。利用提取的E.coliK12基因组DNA为模版,进行PCR扩增。经琼脂糖凝胶电泳验证,电泳条带约650bp与nfsB基因大小一致。使用TaKaRaAgaroseGelDNAPurificationKitVer.3.0切胶回收上述电泳目的条带,然后用EcoRI/NdeI双酶切处理回收产物,使用TaKaRaDNAFragmentPurificationKitVer.3.0将其纯化,之后使用DNALigationKitVer.3.0中的连接酶,将纯化后得到的目的产物与经EcoRI/NdeI双酶切处理的pET28a(+)空载体(Invitrogen)连接后,热激转化至E.coliDH5α(TaKaRa)感受态细胞中,涂布平板,37℃过夜培养。挑选阳性菌落过夜37℃培养后提取质粒并送TaKaRa测序确认与nfsB基因(GeneID:945778)序列一致,验证基因克隆成功,将其命名为野生型nfsB基因,并将其对应的阳性菌落和重组质粒分别命名为克隆菌E.coliDH5α-nfsB-WT和质粒pET28a-nfsB-WT。
2.突变型NfsBF123A的基因获取及表达载体构建
过夜培养E.coliDH5α-nfsB-WT克隆菌,提取pET28a-nfsB-WT重组质粒,以此为模板构建突变体载体pET28a-nfsB-F123A。
首先以质粒pET28a-nfsB-WT为模板,使用引物nfsB-WT-F和nfsB-F123A-R:5’-ATATCAGCGAAGGCCTTGCGACCTTTATCGTTCG-3’进行一次PCR反应,命名为nfsB-F123A-1。同样以质粒pET28a-nfsB-WT为模板,使用引物nfsB-WT-R和nfsB-F123A-F:5’-CGCAAGGCCTTCGCTGATATGCACCGTAAAGATC-3’进行二次PCR反应,命名为nfsB-F123A-2。其中引物nfsB-F123A-F和nfsB-F123A-R中下划线标记的为123位氨基酸突变位点对应的基因序列。使用TaKaRaMiniBestAgaroseGelDNAExtractionKitVer.3.0,对于两次PCR产物切胶回收纯化。以回收产物nfsB-F123A-1和nfsB-F123A-2作为模板,使用引物nfsB-WT-F和nfsB-F123A-R进行第三次重叠延伸PCR,PCR产物使用TaKaRaDNAFragmentPurificationKitVer.3.0回收纯化,获得目的基因,其命名为nfsB-F123A基因,其序列信息为SEQIDNO:1。
将获得的突变体nfsB-F123A基因SEQIDNO:1和pET28a(+)空载使用NdeⅠ/EcoRⅠ分别进行双酶切,使用TaKaRaAgaroseGelDNAPurificationKitVer.3.0切胶回收上述电泳目的条带,之后使用DNALigationKitVer.3.0中的连接酶,将纯化后得到的目的产物与经EcoRI/NdeI双酶切处理的pET28a(+)载体连接后,热激转化至E.coliDH5α感受态细胞中,涂布平板,37℃过夜培养。挑选阳性菌落过夜37℃培养后提取质粒并送TaKaRa测序确认与SEQIDNO:1序列一致,验证表达载体构建成功,并将其对应的阳性菌落和重组质粒命名为克隆菌E.coliDH5α-nfsB-F123A和质粒pET28a-nfsB-F123A。
实施例2.细菌硝基还原酶突变体NfsB-F123A的表达纯化
1.硝基还原酶突变体NfsB-F123A的诱导表达
将重组质粒pET28a-nfsB-F123A热激转化到大肠杆菌E.coliBL21(DE3)感受态细胞(Invitrogen),涂布平板,37℃过夜培养。挑取阳性克隆,并将其命名为表达菌E.coliBL21(DE3)-nfsB-F123A,再接种于LB液体培养基中(含50μg/mlKana),37℃振荡过夜。将种子培养液以1%接种量转接至100mlLB扩大培养基中(含50μg/mlKana),37℃摇床培养至OD600在0.4-0.6。加入终浓度为0.5mMIPTG于30℃下诱导表达6h。培养结束后,发酵液于5000r/min离心10min收集菌体。
2.硝基还原酶突变体NfsB-F123A的纯化
收集菌体用20mM磷酸盐缓冲液(pH7.4,含有500mMNaCl)进行重悬,利用高压匀浆破碎仪在低温下进行破碎,12000r/min离心5min收集上清。
样品分离纯化均使用蛋白质快速层析系统(GEHealthcare)完成,采用GEHealthcareFFHisTrap层析柱(即Ni柱)。首先用缓冲液A(20mMNaH2PO4,0.5MNaCl,pH7.4)平衡Ni柱,将破碎后收集的蛋白上清样品以1ml/min的流速通过Ni柱,使目的蛋白结合于Ni柱。待紫外基线平稳后,使用缓冲液B(elutionbuffer:20mMNaH2PO4,0.5MNaCl,250mM咪唑,pH7.4)洗脱,洗脱梯度依次为20mM,100mM,250mM咪唑,每个洗脱梯度洗脱体积为20个柱体积。分管收集每个梯度洗脱的流出液,对每管样品分别进行蛋白质含量、活力以及SDS-PAGE分析。
3.硝基还原酶突变体NfsB-F123A的SDS-PAGE电泳分析及Western-Blotting验证
利用SDS-PAGE电泳分析突变型硝基还原酶F123A的分子量及其纯度,其试验数据如图2所示,对照Marker选用低分子量标准蛋白。待电泳结束后,用考马斯亮蓝R-250染色3小时以上,用脱色液(以乙醇:醋酸:水的体积比为1:2:17配制而成)震荡脱色,观察电泳结果。
电泳结果发现在分子量27kDa左右有一条明显的诱导条带。同时诱导条带对应蛋白在250mM咪唑洗脱组分得到富集,初步判断该蛋白为突变体蛋白NfsB-F123A,其序列信息为SEQIDNO:2。
针对转膜仪,采用半干法转膜,转膜缓冲液为48mMTris,39mM甘氨酸,20%甲醇,0.037%SDS,具体步骤如下:
将SDS聚丙烯酰胺凝胶取下,置于转膜缓冲液中平衡,剪裁一块大小相同的PVDF膜,将其放置于100%甲醇中激活10s,然后将膜用去离子水清洗掉膜上残余的甲醇,置于转膜缓冲液中;按滤纸-PVDF膜-凝胶-滤纸,自下而上顺序组装,准备转膜;将转膜仪组装完毕,设置恒压18V,转膜2个小时;
封闭:将转完的PVDF膜浸泡在含有5%的脱脂奶粉的TBST缓冲液中(20mMTris-HCl,150mMNaCl,0.05%的Tween20)中封闭1h;
一抗包被:将封闭好的PVDF膜浸泡于含一抗TBST包被液中(含anti-Hisantibody,1:3000,Invitrogen,3%脱脂奶粉)1小时;用TBST缓冲液每隔10min洗涤一次,洗6次,去除非特异性结合抗体;
二抗包被:将上步中的PVDF膜浸泡在含有山羊抗鼠IgG/辣根酶标记抗体和3%的脱脂奶粉的的TBST缓冲液中,振荡1小时;用TBST缓冲液每隔10min洗涤一次,洗6次,,去除非特异性结合的二抗;
用TBST缓冲液每隔10min洗涤一次,洗6次,加入发色底物反应3-5min,曝光,显影,定影。
Western-Blotting结果显示IPTG诱导组分中分子量27kDa处蛋白条带为目的蛋白条带,且该蛋白经Ni柱分离纯化后于250mM咪唑下中得到富集分离,纯度达90%以上。
实施例3.硝基还原酶突变体NfsB-F123A的活性测定
将250mM咪唑洗脱下来的重组蛋白,按照摩尔浓度比蛋白:FMN=1:10的比例混匀,放置4℃冰箱中孵育2个小时。孵育结束后,将重组蛋白脱盐至20mMTris-HCl缓冲液(pH7.0)中,以进行后续蛋白活性检测等实验。
1.HPLC分析硝基还原酶突变体NfsB-F123A的催化活性
用高效液相色谱(HPLC)安捷伦1200分析突变体F123A对芳香硝基底物的还原。以各种芳香硝基化合物为底物,检测突变体的还原效率和还原产物。HPLC分析柱选用C18反相疏水层析柱,粒径5μm,4.6mm×250mm,流动相组成为甲醇和水体系,流速为0.8ml/min,进样体积为10μl,用紫外检测器检测。
HPLC酶催化反应体系中含有:0.1mM芳香硝基底物,0.2mMNADH,50nM突变体酶,反应缓冲液为20mMTris-HClpH7.0,室温反应5-15分钟。
CB1954的HPLC分析:由于CB1954及其反应产物加热不稳定,所以用等体积乙酸乙酯萃取以终止反应,12000×g离心10min。取上层乙酸乙酯相用于HPLC分析。洗脱梯度为20%-60%甲醇线性洗脱30min,检测波长为262nm,柱温20℃。
2,4-DNT的HPLC分析:还原反应终止同TNT。HPLC分析体系为40%-90%的甲醇20min梯度洗脱,检测波长为254nm,柱温20℃。
TNT、2ADNT及4ADNT的HPLC分析:反应结束后,将样品煮沸5min以终止反应,12000×g离心10min,取上清用于HPLC分析。洗脱梯度为30min40%-100%甲醇,检测波长230nm,柱温20℃。
2.硝基还原酶突变体NfsB-F123A的动力学测定
所有的动力学常数都是用全波长扫描荧光酶标仪(ThermoFisherScientific)用连续测点的方法于37℃恒温检测。硝基还原酶动力学检测体系:总体积200μl,20mMTris-HCl(pH7.0)的缓冲液中含有60μMNADH,一系列不同浓度的待测底物和适量的酶。随反应进行,通过检测340nm下NADH的消耗量,进而计算底物消耗反应速率。每还原1mol硝基基团,需要消耗2mol的NADH分子。所有的动力学实验至少重复3次,数据用非线性拟合软件Origin8.5处理得出数据以及误差范围。
表1.硝基还原酶突变体(NfsB-F123A)与野生型蛋白(Wild-type)对不同硝基底物的催化活性比较。
表中,CB1954:5-(1-氮丙啶)-2,4-二硝基苯甲酰胺,一种癌症治疗前体药物;
DNT:2,4-二硝基甲苯,一种重要的工业原料,用于聚氨酯、染料、医药等有机合成中;
TNT:2,4,6-三硝基甲苯,环境污染物的主要成分之一,可导致生物有机体基因突变甚至引发癌症;
2ADNT(2-氨基-4,6-二硝基甲苯)和4ADNT(2-氨基-4,6-二硝基甲苯):环境污染物,较TNT而言具有更高的生物毒性。
由图中数据分析可知,硝基还原酶突变体NfsB-F123A对不同硝基底物的催化活性较野生型而言均有显著的提高。其中突变体NfsB-F123A除对难以降解的污染物TNT活性增加一倍外,对其氨基积累代谢产物(2ADNT和4ADNT)活性提高尤为显著,催化效率达野生型酶的5倍。同时对于重要的芳香硝基原料癌症治疗前药CB1954和DNT而言,催化效率比野生型提高了2.3倍和1.7倍。
Claims (7)
1.一种细菌硝基还原酶突变体基因,其核苷酸序列如SEQIDNO:1所示。
2.如权利要求1所述的细菌硝基还原酶突变体基因所编码的蛋白质,其氨基酸序列如SEQIDNO:2所示。
3.一种包含如权利要求1所述基因的重组表达载体。
4.一种包含如权利要求3所述的重组表达载体的重组表达转化体。
5.一种重组硝基还原酶的制备方法,其特征在于:培养如权利要求4所述的重组表达转化体,从培养物中纯化得到突变型细菌硝基还原酶的蛋白。
6.如权利要求2所述的蛋白质或如权利要求4所述的重组表达转化体作为催化剂在催化硝基还原中的应用。
7.如权利要求2所述的蛋白质或如权利要求4所述的重组表达转化体作为催化剂在药物激活剂代谢、芳香羟胺化合物的生物合成以及硝基类环境污染物生物代谢领域的应用。
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NCBI Reference Sequence: WP_000351487.1;NCBI;《NCBI GenBank》;20130204;全文 * |
新克隆的硝基还原酶基因NOR1的表达及其产物的纯化;聂新民等;《癌症》;20031231;136-139 * |
硝基还原酶/CB1954自杀基因系统对宫颈癌Hela细胞杀伤效应的实验研究;滕隔玲等;《现代肿瘤医学》;20090831;1403-1406 * |
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