CN112301008A - 一种醛还原酶的氨基酸序列及其应用 - Google Patents
一种醛还原酶的氨基酸序列及其应用 Download PDFInfo
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- CN112301008A CN112301008A CN201910673839.4A CN201910673839A CN112301008A CN 112301008 A CN112301008 A CN 112301008A CN 201910673839 A CN201910673839 A CN 201910673839A CN 112301008 A CN112301008 A CN 112301008A
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Abstract
本发明公开了一种醛还原酶的氨基酸序列及其应用,具体公开了一种醛还原酶EupfE,其氨基酸序列如SEQ ID NO.1所示;公开了编码该醛还原酶基因的核苷酸序列,如SEQ ID NO.2所示,及经密码子优化后的核苷酸序列,如SEQ ID NO.3所示,使优化的醛还原酶EupfE的基因可以在大肠杆菌中表达;同时公开了含有所述核苷酸序列的蛋白表达载体和宿主细胞。本发明还公开了醛还原酶EupfE在催化醛类底物stipitaldehyde及其类似物或衍生物发生还原反应形成醇中的应用,该反应可用于抗肿瘤化合物neosetophomone B及其类似物的制备。
Description
技术领域
本发明属于合成生物学、基因工程领域,具体地,本发明涉及一种能够催化 醛类底物发生还原反应形成醇的醛还原酶EupfE的氨基酸序列及其应用。
背景技术
癌症已成为危害人类健康的主要杀手,抗癌药物的研究与开发因而成为新药 研发领域的关注热点。目前,越来越多的来源于动物、植物、真菌、细菌的天然 产物被发现具有突出的抗肿瘤活性,其中已有多个天然来源的抗肿瘤新药已上市 并广泛应用于临床,如紫杉醇、阿糖胞苷等,还有多个天然来源的抗肿瘤活性先 导化合物进入临床试验。然而,许多活性天然产物的含量并不高,富集困难且成 本较高,同时这些天然产物往往结构复杂、具有多个手性中心,全合成往往步骤 多、收率低,或难以工业化,这些因素制约着活性天然产物的进一步研究和开发。 近年来,随着生物技术和基因工程的发展,酶作为绿色、高效、高立体选择性的 催化剂在天然产物及药物合成过程中扮演的角色愈发重要,同时合成生物学手段 也越来越多地被应用于活性天然产物或药物的全合成过程中。
以neosetophomone B为代表的杂萜类化合物是一类真菌次级代谢产物。该类 化合物大多产自微紫青霉菌Penicillium janthinellum,Phoma sp.(J.Zhang,et al,Journal of Natural Products,2015,78,3058-3066),Neosetophoma sp.(T.El-Elimat,et al,Organic Letters,2019,21,529-534),Eupenicillium brefeldianum ATCC 74184(F. Mayerl,et al,Journal of Antibiotics,1993,46,1082-1088),或Kionochaetaramifera BCC 7585(T.Bunyapaiboonsri,et al,Phytochemistry Letters,2008,1,204-206)等。 生源上,该类化合物推测是由humulene类倍半萜衍生物与聚酮产物(tropoloneorthoquinone methides或o-benzoquinone methide)经Diels-Alder反应而成。生物活 性方面,该类化合物绝大多数对肿瘤细胞具有显著的细胞毒作用。其中, neosetophomone B对人卵巢癌OVCAR-8细胞、人肺癌MSTO-211H细胞和大鼠腹 水癌LLC细胞的半数抑制率分别为1.08μM、0.28μM和0.12μM。然而该类化合 物在真菌代谢产物中的含量很低,限制了该类抗肿瘤化合物的进一步研究和开发。 因此,找到该类杂萜化合物在真菌中的生物合成基因簇并对其中的关键蛋白进行 功能鉴定,对于酶催化合成该类杂萜化合物或者通过合成生物学技术规模化制备 该类化合物,减少由于化学合成以及提取分离等传统方法对环境造成的压力,从 而促进社会可持续发展具有重要的意义。
本发明通过提取微紫青霉菌RNA,利用RT-PCR,从微紫青霉菌中克隆得到 了一种编码醛还原酶的eupfE基因,该基因亦可通过合成获得。本发明还鉴定了该 基因编码的蛋白的功能,结果表明该醛还原酶EupfE能够催化以已知化合物stipitaldehyde(AngewandteChemie-International Edition,2014,53,(29):7519-7523) 为代表的化合物中的醛基还原形成相应的醇,从而完成neosetophomone B等杂萜 化合物生物合成的关键步骤。
发明内容
本发明解决的技术问题是提供一种醛还原酶、编码该酶的核苷酸序列、含有 该核苷酸序列的表达载体、含有该核苷酸序列或表达载体的宿主细胞,以及其在 催化醛类底物发生还原反应形成醇的反应中的应用,该反应可用于制备以 neosetophomone B为代表的杂萜类抗肿瘤化合物。所述醛类底物优选为 stipitaldehyde或2,4-二羟基-6-甲基苯甲醛,或它们的类似物或衍生物。
为解决本发明的技术问题,采用如下技术方案:
本发明技术方案的第一方面提供了一种醛还原酶EupfE,其特征在于,其氨基 酸序列为:
(1)序列表中SEQ ID NO.1所示的氨基酸序列;
(2)序列表中SEQ ID NO.1所示的氨基酸序列经替换、缺失或添加1-35个 氨基酸形成的基本上保持相同生物学功能的氨基酸序列。
以上所述的醛还原酶EupfE,其特征在于,在醛还原酶EupfE上可进行常规修 饰;或者在醛还原酶EupfE上还连接有用于检测或纯化的标签。
其中,所述的常规修饰包括乙酰化、酰胺化、环化、糖基化、磷酸化、烷基 化、生物素化、荧光基团修饰、聚乙二醇PEG修饰、固定化修饰;所述的标签包 括His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc、Profinity eXact。
本发明提供的醛还原酶EupfE可以是重组多肽、天然多肽、合成多肽,优选 重组多肽。本发明的多肽可以是天然纯化的产物,或是化学合成的产物,或使用 重组技术从原核或真核宿主(例如,细菌、酵母、高等植物、昆虫和哺乳动物) 细胞中产生。
本发明技术方案的第二方面提供了一种编码第一方面所述醛还原酶EupfE的 核酸分子,其特征在于:
(1)序列表中SEQ ID NO.2所述的核苷酸序列;
(2)基于SEQ ID NO.2所述的核苷酸序列根据大肠杆菌的密码子偏好性进行 序列优化得到的序列表中SEQ ID NO.3所述的核苷酸序列;
(3)与上述(1)或(2)中的核苷酸序列互补的核苷酸序列。
其中,以上(2)中所述的经密码子优化后的核苷酸序列更容易在大肠杆菌中 表达形成可溶性的醛还原酶EupfE。
术语“编码醛还原酶EupfE的核酸分子”是指包括编码醛还原酶EupfE多肽 的核苷酸序列和包括附加编码和/或非编码的核苷酸序列,它包括:只有成熟多肽 的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和 任选的附加编码序列)以及非编码序列。
本发明的核酸分子可以是DNA形式或RNA形式。DNA形式包括cDNA、基 因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码 链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.2或SEQ ID NO.3 所示的编码区序列相同或是简并的变异体。如本发明所用,“简并的变异体”在本 发明中是指编码具有SEQ ID NO.1的蛋白质或多肽,但与SEQ ID NO.2或SEQ ID NO.3所示的编码区序列有差别的核苷酸序列。
通过本文的阐述,这样的片段、类似物或衍生物被认为在本领域技术人员的 知识范围内。
本发明还涉及与以上所描述的序列杂交的核酸片段。如本发明所用,“核酸片 段”的长度至少含10个核苷酸,优选是至少20-30个核苷酸,特别优选是至少50-60 个核苷酸,非常特别的优选是至少100个核苷酸以上。核苷酸片段也可用于核酸 的扩增技术(如PCR)以确定和/或分离编码的核苷酸序列。
本发明中特异核苷酸序列能用多种方法获得。例如,用本领域熟知的杂交技 术分离核苷酸序列。这些技术包括但不局限于:(1)用探针与基因组或cDNA文 库杂交以检出同源的核苷酸序列;和(2)表达文库的抗体筛选以检出具有共同结 构特征的克隆的核苷酸序列片段。
本发明的DNA片段序列也能用下列方法获得:(1)从基因组DNA分离双链 DNA序列;(2)常规化学合成方法合成该DNA序列以获得所述醛还原酶EupfE 的双链DNA。
在上述提到的方法中,分离基因组DNA最不常用。DNA序列的直接化学合 成是经常选用的方法。更经常选用的方法是DNA序列的分离。分离感兴趣的cDNA 的标准方法是从高表达该基因的供体细胞分离mRNA并进行逆转录,形成质粒或 噬菌体cDNA文库。提取mRNA的方法已有多种成熟的技术,用试剂盒也可从商 业途径获得。而构建cDNA文库也是通常的方法。当结合聚合酶链式反应技术 (PCR)时,即使极少的表达产物也能克隆。
可用常规方法从这些cDNA文库中筛选本发明的基因,这些方法包括但不局 限于:(1)DNA-DNA或DNA-RNA杂交;(2)标志基因功能的出现或消失;(3) 测定的转录本的水平;(4)通过免疫学技术测定生物学活性,来检测基因表达的 蛋白产物。上述方法可单用,也可多种方法联合应用。在第(1)种方法中,杂交 所用的探针是与本发明的核苷酸序列的任何一部分同源,其长度至少10个核苷酸, 优选是至少30个核苷酸,特别优选是至少50个核苷酸,非常特别的优选是至少 100个核苷酸。此外,探针的长度通常在2000个核苷酸之内,较佳的为1000个核 苷酸之内。此处所用的探针通常是在本发明的基因序列信息的基础上化学合成的 DNA序列。本发明的基因本身或者片段当然可以用作探针。DNA探针的标记可用放射性同位素,荧光素或酶(如碱性磷酸酶)等。
应用PCR技术扩增DNA/RNA的方法(Saiki et al.,1985,Science,230, 1350-1354)被优选用于获得本发明的基因。特别是很难从文库中得到全长的cDNA 时,可优选使用RACE法(RACE-cDNA末端快速扩增法),用于PCR的引物可根 据本文所公开的本发明的核苷酸序列信息适当的选择,并可用常规方法合成。可 用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明技术方案的第三方面提供了一种含有第二方面所述核酸分子的表达载体。
本发明也涉及含有编码醛还原酶EupfE核苷酸序列和外源性调节序列元件结 合进行功能性表达的重组表达载体。术语“载体”指本领域熟知的细菌质粒、噬菌体、 酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其它载体。 能够影响基因表达产物的序列元件包括有复制起始点、启动子、标记基因和翻译 调控元件。
可用本领域的技术人员熟知的方法来构建含编码醛还原酶EupfE的核苷酸序 列和合适的转录/翻译调控元件的表达载体。这些方法包括体外重组DNA技术、 DNA合成技术、体内重组技术等(Sambroook,et al.,Molecular Cloning,a Laboratory Manual,ColdSpring Harbor Laboratory,New York,1989)。所述的编码醛还原酶 EupfE的核苷酸序列可有效连接到表达载体的恰当启动子上,以指导mRNA合 成。这些启动子的代表性例子有:大肠杆菌的lac或trp启动子;λ噬菌体的PL 启动子;真核启动子包括CMV早期启动子、HSV胸苷激酶启动子、早期和晚期 SV40启动子、反转录病毒的LTRs和其它一些已知的可控制基因在原核细胞或真 核细胞或病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和 转录终止子等。在载体中插入增强子序列将会使其在高等真核细胞中的转录得到 增强。增强子是DNA表达的顺式作用因子,通常大约有10-300bp,作用于启动 子以增强基因的转录。如腺病毒增强子。
本发明技术方案的第四方面提供了一种含有第三方面所述表达载体的宿主细胞,其中所述宿主细胞选自细菌,酵母,曲霉菌,植物细胞,昆虫细胞;优选为 大肠杆菌或酵母菌,所述酵母菌包括但不限于毕赤酵母,酿酒酵母等;更优选的 宿主细胞为大肠杆菌。
本发明涉及用含有本发明编码醛还原酶EupfE的核苷酸序列的重组载体或直 接用编码醛还原酶EupfE的核苷酸序列经基因工程产生的宿主细胞。
本发明中,编码醛还原酶EupfE的核苷酸序列或含有该核苷酸序列的重组表 达载体可转化或转导入宿主细胞,以构成含有该核苷酸序列或重组表达载体的基 因工程化宿主细胞。术语“宿主细胞”指原核细胞,如细菌细胞;或是低等真核细胞, 如酵母细胞;或是高等真核细胞,如哺乳动物细胞。宿主细胞的代表性例子有: 原核细胞如大肠杆菌;真菌细胞如酵母;植物细胞如油菜、烟草、大豆;昆虫细 胞如果蝇S2或Sf9;动物细胞如CHO、COS或Bowes黑素瘤细胞等。
用本发明所述的核苷酸序列或含有核苷酸序列的重组表达载体转化宿主细胞 可用本领域的技术人员熟知的方法进行。当宿主为原核生物如大肠杆菌时,能吸 收DNA的感受态细胞可在指数生长期收集菌体,用CaCl2法处理,所用的步骤在 本领域是众所周知的。也可用MgCl2,电穿孔等方法进行。当宿主是真核生物,可 选用DNA转染法、显微注射、电穿孔、脂质体包装等方法。
本发明技术方案的第五方面提供了第一方面所述的醛还原酶EupfE或第二方 面所述核苷酸分子或第三方面所述表达载体或第四方面所述宿主细胞在催化底物 中的醛基还原成醇中的应用。
以上所述的醛还原酶EupfE催化的底物为以下通式(I)或通式(II)所代表 的化合物。
其中:
通式(I)和(II)中X可选自O、N、C或S原子;优选为O或N原子;最 优选为O原子。
通式(I)中的R1和通式(II)中的R7各自独立选自氢、C1-4烷基、C1-4羟烷 基、C2-4烯基、乙酰基、丙酰基、丁酰基、异丁酰基、苯甲酰基、苯乙烯酰基、苄 基或卤代苄基;优选为氢或C1-4烷基,更优选为氢或甲基,最优选为氢。
通式(I)中的R2与R4以及通式(II)的R6与R8各自独立选自氢、羟基、卤 素、C1-4烷氧基、苄氧基、苯甲酰氧基或苯乙烯酰氧基;优选为氢或C1-4烷基,更 优选为氢或甲基,最优选为氢。
通式(I)中的R3和通式(II)中的R5各自独立选自氢、C1-4烷基、C1-4羟烷 基、卤代的C1-4烷基、C2-4烯基或卤代C2-4烯基;优选为氢或C1-4烷基,更优选为 氢或甲基,最优选为甲基。
以上所述的醛还原酶EupfE催化的底物优选为stipitaldehyde或2,4-二羟基-6-甲基苯甲醛。
该方法是这样实现的,根据宿主细胞,用本领域技术人员所共知的方法生长 或培养。比如微生物细胞通常是在0-100℃,优选10-60℃,同时还要氧气。培养 基中含有碳源,如葡萄糖;氮源,通常是有机氮的形式,如酵母提取物、氨基酸; 盐,如硫酸铵;微量元素,如铁、镁盐;如果需要的话还有维生素。在此期间培 养基的pH可以保持固定的值,就是说,在培养期间进行控制或不控制。培养可以 分批培养、半不连续培养或连续培养形式进行。在培养之后,收集细胞,破碎或 直接使用。可将通式(I)或(II)的底物与上述破碎后的宿主细胞在缓冲溶液中 孵育,所述缓冲溶液包括但不限于磷酸盐缓冲液(PBS)、三羟甲基胺基甲烷-盐酸 缓冲液(Tris-HCl)、N,N-二羟乙基甘氨酸缓冲液(Bicine)、2-吗啉代乙磺酸缓冲液(MES)、4-羟乙基哌嗪乙磺酸缓冲液(HEPES)等,孵育温度为10-40℃,优 选20-30℃,必要时需加入辅因子,所述辅因子优选为还原型烟酰胺腺嘌呤二核苷 酸磷酸(又称还原型辅酶II,NADPH)或还原型烟酰胺腺嘌呤二核苷酸(又称还 原型辅酶I,NADH),即可获得底物中醛基还原为醇的产物。亦可将通式(I)或 (II)的底物喂养至上述宿主细胞,在10-50℃,优选25-37℃,培养1-10天(原 核宿主优选1-2天,真核宿主优选3-7天),破碎或不破碎细胞,加入有机溶剂萃 取,即可获得底物中醛基还原为醇的产物。还可将通式(I)或(II)的底物与第 一方面所述的醛还原酶EupfE在缓冲溶液中孵育,所述缓冲溶液包括但不限于磷 酸盐缓冲液(PBS)、三羟甲基胺基甲烷-盐酸缓冲液(Tris-HCl)、N,N-二羟乙基甘 氨酸缓冲液(Bicine)、2-吗啉代乙磺酸缓冲液(MES)、4-羟乙基哌嗪乙磺酸缓冲 液(HEPES)等,孵育温度为10-40℃,优选20-30℃,必要时需加入辅因子,所 述辅因子优选为NADPH或NADH,即可获得底物中醛基还原为醇的产物。
本发明技术方案的第六方面提供了第五方面所述的由醛基还原形成-CH2OH 的反应产物在制备抗肿瘤化合物neosetophomone B及其类似物中的应用。
本发明的其他方面由于本文技术的公开,对本领域的技术人员而言是显而易 见的。
本发明的有益技术效果:以neosetophomone B,pughiinin A为代表的杂萜类 化合物是一类由真菌Penicillium janthinellum,Kionochaeta pughii,Phoma sp.(J.Zhang,et al,J.Nat.Prod.,2015,78,3058-3066)等产生的次级代谢产物,大多具有 显著的抗肿瘤作用。然而该类化合物在真菌代谢产物中的含量低,目前尚无全合 成研究报道,限制了其进一步的研究和开发,给相关创新药物的研制带来了困难。 本发明技术方案提供的醛还原酶EupfE能够在温和条件下高效催化通式(I)或通 式(II)所代表的化合物中的醛基还原形成-CH2OH,该还原反应的产物是多种抗 肿瘤杂萜类化合物合成过程中的重要中间体,解决了通过合成生物学手段合成以 neosetophomone B为代表的杂萜类化合物及其类似物的关键技术难题。
附图说明
图1:eupfE基因PCR产物1%琼脂糖凝胶电泳分析结果,其中M是DNA分 子量标准。
图2:重组质粒pET28a-eupfE示意图。
图3:EupfE重组蛋白SDS-PAGE分析结果,其中M为蛋白分子量标准。
图4:EupfE在辅因子NADPH或NADH的存在下对stipitaldehyde的还原作 用。
图5:Stipitol的1H NMR谱图(C6D6:DMSO-d6=4:1,600MHz)。
图6:Stipitol的13C NMR谱图(C6D6:DMSO-d6=4:1,150MHz)。
图7:Stipitol的HSQC谱图(C6D6:DMSO-d6=4:1,600MHz)。
图8:Stipitol的HMBC谱图(C6D6:DMSO-d6=4:1,600MHz)。
图9:EupfE对底物A1的还原作用。
图10:EupfE对底物B1的还原作用。
具体实施方式
本发明通过如下实施例进行进一步的说明,这些实施例仅用于说明性的,而 不是以任何方式限制本发明权利要求的范围。
实施例1.eupfE基因的获取及表达载体的构建
将微紫青霉菌P.janthinellum接种至马铃薯葡萄糖培养基(PDB)中,于28℃ 摇床(220rpm)培养4天,取少量菌丝体于研钵中加入液氮研磨成细粉状。采用 Trizol提取法提取微紫青霉菌总RNA,总RNA经RQ1RNase-Free DNase消化DNA 获得无DNA污染的总RNA。采用RT-PCR法将纯化后的总RNA反转录成cDNA。 反转录体系和程序如下:20μL的反应体系中包含总RNA 13μL,50μM Oligo dT Primer 1μL,100μM Random 6mers 1μL,5×PrimeScriptbuffer 4μL,PrimeScript RT enzyme Mix 1μL;该体系在37℃温育30min后85℃变性5sec,完成cDNA 的合成。cDNA保存于-20℃备用。
以上述微紫青霉菌cDNA为模板,设计两条特异引物(正向引物: 5’-ATGGGATCACTCGACTCTAAACC-3’,SEQ ID NO.4;反向引物: 5’-TTACCACGGCTGTACCTCATC-3’,SEQ ID NO.5),通过PCR克隆去内含子 eupfE基因。PCR体系(50μL)中含5×Q5buffer 10μL,10mM dNTPs 1μL,GC Enhancer 5μL,10μM的正向引物(SEQ ID NO.4)和10μM的反向引物(SEQ ID NO.5)各2.5μL,cDNA 2μL,Q5DNA聚合酶0.5μL,双蒸水26.5μL。PCR程 序:预变性:98℃,1min;循环:98℃变性10sec,57℃复性30sec,72℃延伸 1min,共34个循环;终延伸:72℃延伸5min,12℃保温。1%琼脂糖凝胶电泳 分析PCR产物,结果表明,扩增得到一条长约为837bp大小的条带(图1)。将该 PCR产物进行DNA测序,表明其序列为SEQ ID NO.2所述DNA序列。将该DNA 序列连接于pET-28a(+)蛋白表达载体上,得到相应表达质粒。或者将SEQ IDNO.2 所述的DNA序列经本领域技术人员熟知的方法进行化学合成,并连接于pET-28a(+)蛋白表达载体上,得到相应质粒。此外,可根据大肠杆菌的密码子偏好性进行序 列优化得到序列表中SEQ ID NO.3所述的DNA序列,该序列经本领域技术人员熟 知的方法进行全合成并连接于pET-28a(+)蛋白表达载体上,得到pET28a-eupfE质 粒(图2)。
实施例2.重组EupfE蛋白的诱导表达和纯化
将构建好的质粒pET28a-eupfE用热激法转化至蛋白表达宿主BL21(DE3)大 肠杆菌,转化菌涂布于含卡那霉素(终浓度50μg/mL)的Luria-Bertani(LB)固 体培养基(10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,15g/L琼脂粉) 上,于37℃培养过夜,直至长出单克隆。挑取单克隆转接入5mL含卡那霉素(终 浓度50μg/mL)的LB液体培养基(10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯 化钠),37℃,220rpm培养过夜以获得种子液。取1mL种子液接入1L含卡那霉 素(终浓度50μg/mL)的LB液体培养基,37℃,220rpm培养至OD600为0.55 时,加入终浓度为50μM的IPTG,在16℃,220rpm诱导20小时。菌液于4℃, 4000rpm离心10min,弃上清,加入50mL预冷裂解缓冲液(50mM Tris-HCl, pH 8.0,10mM咪唑,150mM氯化钠,10%甘油)重悬菌体,低温高压破碎。破 碎液于4℃,14000rpm离心20min,吸取上清为EupfE粗蛋白。粗蛋白采用Ni-NTA 亲和层析柱纯化,并用Ultra-15 10K超滤管浓缩并替换至Buffer C(50mM Tris-HCl,pH 8.0,150mM氯化钠,10%甘油),测定蛋白浓度。EupfE的SDS-PAGE 分析结果如图3所示,表达纯化得到一条长约为34kDa的特异性条带,与EupfE 的理论大小一致。
实施例3.EupfE在辅因子NADPH存在下的催化活性
本实验以已知化合物stipitaldehyde为底物,探讨醛还原酶EupfE对该底物的 还原作用,结果表明,EupfE能催化相应底物中的醛还原形成醇。
为验证醛还原酶EupfE的催化功能,建立100μL反应体系(含50mM Bicine 缓冲液,pH 8.0,2mM stipitaldehyde,3mM NADPH和25μM EupfE),26℃反应 20min后,加入100μL乙酸乙酯终止反应。将乙酸乙酯萃取物浓缩干燥后用乙腈 溶解,液-质联用仪分析。流动相为乙腈-水(均加入0.02%甲酸),流速0.4mL/min, 梯度洗脱条件:0-8min,5-99%乙腈;8-12min,99%乙腈。液质联用仪结果(图 4)表明,醛还原酶EupfE在辅因子NADPH的存在下能够催化stipitaldehyde反应 形成stipitol。Stipitol的结构通过1H NMR(图5)、13C NMR(图6)、HSQC(图 7)和HMBC(图8)谱图,并与stipitaldehyde的谱图进行对比(表1)进行鉴定。
表1.EupfE催化的反应产物stipitol的NMR数据(与stipitaldehyde对比)
a测定溶剂为C6D6-(CD3)2SO(4:1,v/v);1H NMR为600MHz,13C NMR为150MHz;
b测定溶剂为CD3COCD3;1H NMR为600MHz,13C NMR为150MHz。
实施例4.EupfE在辅因子NADH存在下的催化活性
本实验以stipitaldehyde为底物,探讨醛还原酶EupfE在辅因子NADH存在下 对该底物的还原作用。
首先,建立100μL反应体系(含50mM Bicine缓冲液,pH 8.0,2mM stipitaldehyde,3mM NADH和25μM EupfE),26℃反应20min后,加入100μL 乙酸乙酯终止反应。将乙酸乙酯萃取物浓缩干燥后用乙腈溶解,13000rpm离心3 min后采用液-质联用仪分析。流动相为乙腈-水(均加入0.02%甲酸),流速0.4 mL/min,梯度洗脱条件:0-8min,5-99%乙腈;8-12min,99%乙腈。经液质联用 仪分析(图4),表明醛还原酶EupfE可在辅因子NADH的存在下催化底物 stipitaldehyde反应形成stipitol。
实施例5.EupfE在体内的催化活性
本实验通过将底物stipitaldehyde喂养至含有醛还原酶EupfE的宿主细胞,探 讨醛还原酶EupfE在体内对该底物的还原作用。
将构建好的携带质粒pET28a-eupfE的大肠杆菌接种至25mL含卡那霉素(终 浓度50μg/mL)和2mM stipitaldehyde的LB液体培养基(10g/L胰蛋白胨,5g/L 酵母提取物,10g/L氯化钠),于37℃,220rpm培养1天。加入等体积乙酸乙酯 萃取,萃取物浓缩干燥后用乙腈溶解,13000rpm离心3min后采用液-质联用仪分 析。流动相为乙腈-水(均加入0.02%甲酸),流速0.4mL/min,梯度洗脱条件:0-8 min,5-99%乙腈;8-12min,99%乙腈。经液质联用仪分析,表明底物stipitaldehyde 可在宿主体内被还原形成stipitol。
实施例6.EupfE对底物o-orsellinaldehyde的还原作用
本实验以化合物o-orsellinaldehyde(代号A1)为底物(图9),探讨醛还原酶EupfE对该底物的还原作用。
首先,建立100μL反应体系(含50mM Bicine缓冲液,pH 8.0,2mM化合 物A1,3mMNADPH和25μM EupfE),26℃反应20min后,加入100μL乙酸乙 酯终止反应。将乙酸乙酯萃取物浓缩干燥后采用液质联用仪分析,结果表明,醛 还原酶EupfE在辅因子NADPH的存在下能够催化化合物A1反应形成化合物A2 (m/z 155[M+H]+)(图9)。
实施例7.EupfE对底物3-chloro-4,6-dihydroxy-2-methylbenzaldehyde的还原作用
本实验以化合物3-chloro-4,6-dihydroxy-2-methylbenzaldehyde(代号B1)为底物(图10),探讨醛还原酶EupfE对该底物的还原作用。
首先,建立100μL反应体系(含50mM Bicine缓冲液,pH 8.0,2mM化合 物B1,3mMNADPH和25μM EupfE),26℃反应20min后,加入100μL乙酸乙 酯终止反应。将乙酸乙酯萃取物浓缩干燥后采用液质联用仪分析,结果表明,醛 还原酶EupfE在辅因子NADPH的存在下能够催化化合物B1反应形成化合物B2 (m/z 189[M+H]+)(图10)。
序列表
<110> 中国医学科学院药物研究所
<120> 一种醛还原酶的氨基酸序列及其应用
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<170> SIPOSequenceListing 1.0
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<213> 微紫青霉菌(Penicillium janthinellum)
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Met Gly Ser Leu Asp Ser Lys Pro Ile Ile Leu Ile Thr Gly Ala Asn
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Thr Gly Leu Gly Phe Glu Val Ala Lys Thr Leu Phe Ala Arg Pro Asp
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Pro Cys His Ile Leu Ile Gly Cys Arg Gly Asp Ile Ser Arg Ala Ser
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Asp Ala Ile Ser Lys Leu Gln Gln Leu Leu Pro Asp Asn Ser Ser Thr
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Ala Glu Pro Leu Ser Ile Asp Ile Thr Ser Glu Glu Ser Ile Ala Thr
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Ala Phe Lys Gln Val Gln Glu Lys Phe Gly His Val Asp Ile Leu Val
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Asn Asn Ala Gly Leu Ala Leu Asp Thr Ala Val Thr Ser Gly Gln Leu
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Ser Pro Arg Glu Gly Trp Asn Gln Thr Tyr Asn Val Asn Val Thr Gly
115 120 125
Thr His Leu Phe Thr Ser Ala Phe Val Pro Leu Leu Leu Ala Ser Gln
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Ala Gln Thr Lys Arg Leu Ile Phe Ile Thr Ser Gly Leu Ser Ser Ile
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Thr Glu His Ala Ser Gly Ile Ser Pro Arg Tyr Thr Leu Ala Pro Ala
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Gly Trp Pro Lys Pro Asp Thr Leu Trp Phe Pro Tyr Arg Val Ser Lys
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Val Ala Met Asn Met Leu Ala Ala Glu Trp Ala Arg Leu Leu Arg Asn
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Asp Gly Val Val Val Phe Asn Val Ser Pro Gly Phe Leu Asn Thr Gly
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Leu Gly Asp Asn Arg Val Thr Gly Glu Trp Arg Asp Lys Ser Val Met
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Gly Ala Ile Asp Pro Ala Ile Gly Ala Ala Phe Cys Val Asp Val Ile
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<213> 微紫青霉菌(Penicillium janthinellum)
<400> 2
atgggatcac tcgactctaa acccattatt cttatcacgg gtgccaatac tggtctagga 60
ttcgaagtag caaaaactct ctttgcccgc cccgacccct gccacatcct catcggatgt 120
cggggtgata ttagtcgcgc aagcgacgcc atatccaagt tgcaacaact tttgcccgac 180
aactcatcca ccgcagagcc actttccatt gatattacgt cagaagagtc aattgctact 240
gctttcaagc aagtccagga aaaattcggc catgttgaca tcctggtgaa caatgccggc 300
ctagccctag acacagcggt cacctcgggc caactcagcc cacgtgaggg ctggaaccag 360
acatacaacg tcaatgtcac cggcacccac ctcttcactt ccgcctttgt gcccctcctg 420
ctcgcctcgc aggcgcagac caagcgcctg attttcatca ccagtggctt gtcgtccatc 480
acggagcacg caagtggtat aagcccgcgc tacaccctcg cgccggcggg ctggcctaag 540
ccggataccc tatggttccc ctaccgcgtg agtaaggtgg cgatgaacat gctggcggcc 600
gagtgggcgc gcctacttcg caatgacgga gttgttgtgt ttaacgtgtc gccagggttt 660
ttgaacactg ggttgggaga taaccgggtg acgggcgagt ggagggacaa gagtgtcatg 720
ggggctattg atccggccat tggggccgct ttctgtgttg atgttattaa ggggaagagg 780
gatgagcagg cctggcctcc taaggtgatt cggaaggatg aggtacagcc gtggtaa 837
<210> 3
<211> 837
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgggtagcc tggacagcaa gccgatcatt ctgatcaccg gtgcgaacac cggtctgggt 60
tttgaggttg cgaaaaccct gtttgcgcgt ccggacccgt gccacatcct gattggttgc 120
cgtggcgaca tcagccgtgc gagcgatgcg attagcaagc tgcagcaact gctgccggac 180
aacagcagca ccgcggagcc gctgagcatc gatattacca gcgaggaaag catcgcgacc 240
gcgttcaagc aggttcaaga aaaatttggt cacgttgaca ttctggtgaa caacgcgggt 300
ctggcgctgg ataccgcggt taccagcggt cagctgagcc cgcgtgaggg ctggaaccaa 360
acctacaacg ttaacgtgac cggtacccac ctgttcacca gcgcgtttgt tccgctgctg 420
ctggcgagcc aggcgcaaac caagcgtctg atcttcatta ccagcggtct gagcagcatc 480
accgaacacg cgagcggcat tagcccgcgt tacaccctgg cgccggcggg ttggccgaag 540
ccggacaccc tgtggtttcc gtatcgtgtt agcaaagtgg cgatgaacat gctggcggcg 600
gagtgggcgc gtctgctgcg taacgacggt gtggttgtgt tcaacgtgag cccgggcttt 660
ctgaacaccg gtctgggcga taaccgtgtt accggtgaat ggcgtgacaa aagcgtgatg 720
ggtgcgattg atccggcgat tggtgcggcg ttctgcgttg acgtgatcaa gggcaaacgt 780
gatgagcagg cgtggccgcc aaaggttatt cgtaaagatg aagtgcaacc gtggtaa 837
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgggatcac tcgactctaa acc 23
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttaccacggc tgtacctcat c 21
Claims (17)
1.一种醛还原酶EupfE,其特征在于,其氨基酸序列为:
(1)序列表中SEQ ID NO.1所示的氨基酸序列;
(2)序列表中SEQ ID NO.1所示的氨基酸序列经替换、缺失或添加1-35个氨基酸形成的基本上保持相同生物学功能的氨基酸序列。
2.根据权利要求1所述的醛还原酶EupfE,其特征在于,在醛还原酶EupfE上可进行常规修饰;或者在醛还原酶EupfE上还连接有用于检测或纯化的标签。
3.根据权利要求2所述的醛还原酶EupfE的修饰,其特征在于,所述的常规修饰包括乙酰化、酰胺化、环化、糖基化、磷酸化、烷基化、生物素化、荧光基团修饰、聚乙二醇PEG修饰、固定化修饰;所述的标签包括His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc、ProfinityeXact。
4.一种编码权利要求1所述醛还原酶EupfE的核酸分子。
5.根据权利要求4所述的核酸分子,其特征在于:
(1)序列表中SEQ ID NO.2所述的核苷酸序列;
(2)基于SEQ ID NO.2所述的核苷酸序列根据大肠杆菌的密码子偏好性进行序列优化得到的序列表中SEQ ID NO.3所述的核苷酸序列;
(3)与上述(1)或(2)中的核苷酸序列互补的核苷酸序列。
6.一种含有权利要求4-5任一所述核酸分子的表达载体。
7.一种含有权利要求4-5任一所述核酸分子或权利要求6所述表达载体的宿主细胞。
8.根据权利要求7所述的宿主细胞,其特征在于,其宿主细胞选自细菌,酵母,曲霉菌,植物细胞,或昆虫细胞。
9.权利要求1-3任一项所述的醛还原酶EupfE或权利要求4-5任一项所述的核酸分子或权利要求6所述的表达载体或权利要求7-8任一项所述的宿主细胞在催化醛类底物还原成醇中的应用。
10.根据权利要求9所述的应用,其特征在于,所述醛类底物为以下通式(I)或通式(II)所代表的化合物;
其中:
通式(I)和(II)中X为O、N、C或S原子;
通式(I)中的R1和通式(II)中的R7各自独立选自氢、C1-4烷基、C1-4羟烷基、C2-4烯基、乙酰基、丙酰基、丁酰基、异丁酰基、苯甲酰基、苯乙烯酰基、苄基或卤代苄基;
通式(I)中的R2与R4以及通式(II)的R6与R8各自独立选自氢、羟基、卤素、C1-4烷氧基、苄氧基、苯甲酰氧基或苯乙烯酰氧基;
通式(I)中的R3和通式(II)中的R5各自独立选自氢、C1-4烷基、C1-4羟烷基、卤代的C1-4烷基、C2-4烯基或卤代C2-4烯基。
11.根据权利要求10所述的应用,其特征在于,通式(I)中的X为O,且R1、R2、R4均为氢,R3为甲基。
12.根据权利要求10所述的应用,其特征在于,通式(II)中的X为O,且R6、R7、R8均为氢,R5为甲基。
13.根据权利要求9所述的应用,其特征在于,所述的醛类底物为stipitaldehyde及其类似物或衍生物。
14.根据权利要求9所述的应用,其特征在于,所述的醛类底物为2,4-二羟基-6-甲基苯甲醛及其类似物或衍生物。
15.根据权利要求10所述的应用,其特征在于,该催化反应可使用辅因子。
16.根据权利要求15所述的应用,其特征在于,所述辅因子为NADPH或NADH。
17.权利要求1所述的醛还原酶EupfE在制备抗肿瘤化合物neosetophomone B及其类似物中的应用。
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