CN114875004A - 一种高立体选择性r转酮酶突变体及其编码基因和应用 - Google Patents
一种高立体选择性r转酮酶突变体及其编码基因和应用 Download PDFInfo
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- CN114875004A CN114875004A CN202110162619.2A CN202110162619A CN114875004A CN 114875004 A CN114875004 A CN 114875004A CN 202110162619 A CN202110162619 A CN 202110162619A CN 114875004 A CN114875004 A CN 114875004A
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- transketolase
- mutant
- high stereoselectivity
- stereoselectivity
- ala
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Abstract
本发明公开了一种高立体选择性R转酮酶突变体及其编码基因和应用,涉及酶工程技术领域,一种高立体选择性R转酮酶突变体的氨基酸序列为SEQ ID No.1,其编码基因的核苷酸序列为SEQ ID No.2;含有该编码基因的重组表达载体;含有该重组表达载体的宿主细胞;制备该高立体选择性R转酮酶突变体的方法;该R转酮酶突变体在催化芳香醛形成高立体选择性的R‑芳香二羟酮中的应用;其编码基因在催化芳香醛形成高立体选择性的R‑芳香二羟酮中的应用。突变体EcTK1_YYH催化产生的对甲砜基苯基二羟酮的立体选择性为95.2%ee(R),具有高活性和高立体选择性。
Description
技术领域
本发明涉及酶工程技术领域,尤其涉及一种高立体选择性R转酮酶突变体及其编码基因和应用。
背景技术
转酮酶是一种广泛存在的焦磷酸硫胺素依赖的酶。它连接了非氧化的磷酸戊糖途径和三羧酸循环。转酮酶催化可逆的转酮反应,将酮醇供体的二碳单位转移到醛受体中,形成手性的二羟酮类化合物。在转酮反应中,β-羟基丙酮酸通常用作酮醇供体,因为β-羟基丙酮酸被转酮后生成挥发性的二氧化碳,从而使反应不可逆。近来,文献报道了二羟酮化合物的检测方法,包括比色法和HPLC法,并建立了手性GC和HPLC法来检测二羟酮化合物的对映选择性。这些检测方法的建立有利于改造转酮酶突变体的催化活性和立体选择性。
立体选择性以对映体过剩率ee来反映,ee表示一种手性化合物的光学纯度,ee值越高,光学纯度也越高。2008年Helen C.Hailes课题组报道野生型的大肠杆菌转酮酶能催化产生L-赤藓酮糖,其立体选择性较高,为95%ee(S)对映体过剩率用来表示一种手性化合物的光学纯度。ee值越高,光学纯度也越高,但催化戊醛产生中等立体选择性的羟酮产物。有研究通过对转酮酶D469和H26位点的定点突变,提高并反转了转酮酶催化戊醛的立体选择性。其中,D469E的S选择性得到了提高(ee(S)=90%),而H26Y突变体催化产生(R)-羟酮(ee=88%)[Enhancing and Reversing the Stereoselectivity of Escherichia coliTransketolase via Single-Point Mutations,Adv.Synth.Catal.2008,350,2631–2638]。Paul A.Dalby课题组在2019年报道通过对大肠杆菌来源的转酮酶进行饱和突变和组合突变,筛选所得突变体能催化丙酮酸供体。当戊醛和己醛作为受体时,产生高立体选择性的(S)-羟酮(ee>98%)[Engineering transketolase to accept both unnatural donorand acceptor substrates and produce α-hydroxyketones,The FEBS Journal 287(2020)1758–1776]。综上,转酮酶对脂肪醛(比如丙醛,戊醛和己醛)具有较高的催化活性,并通过酶进化获取的转酮酶突变体具有较高的S选择性(ee=90%-98%)和R选择性(ee=88%)。
除了催化脂肪醛,转酮酶被改造后还可以催化芳香醛。2010年Helen C.Hailes课题组报道对大肠杆菌来源的转酮酶进行了D469和F434的定点突变,实现了对苯甲醛和间羟基苯甲醛的催化,但催化效率较低,转化率≤10%。其突变体催化产生的羟酮产物主要为R构型(ee≤82%)[α,α′-Dihydroxyketone formation using aromatic andheteroaromatic aldehydes with evolved transketolase enzymes,Chem.Commun.,2010,46,7608–7610]。Paul A.Dalby课题组2015年报道在大肠杆菌TK/D469T的基础上作R520和S385的迭代饱和突变,筛选所得突变体EcTK/D469T/R520Q/S385Y对苯甲醛衍生物包括间羧基苯甲醛,间羟基苯甲醛和对羧基苯甲醛均具有较高的催化活性,拓宽了转酮酶的底物谱,但其立体选择性未知[Second generation engineering of transketolase forpolar aromatic aldehyde substrates,Enzyme and Microbial TecH26ology 71(2015)45–52]。Wolf-Dieter Fessner课题组2017年报道通过对嗜热脂肪芽孢杆菌来源的转酮酶定向进化,提高了转酮酶对苯基乙醛,苯基丙醛,苯氧基乙醛,苄氧基乙醛的催化效率,产率为60-72%,其羟酮产物具有绝对的S立体选择性(ee>99%)。另外,定向进化所得的转酮酶突变体L382N/D470S对苯甲醛底物具有良好的催化活性,但未报道其立体选择性[Secondgeneration engineering of transketolase for polar aromatic aldehydesubstrates,Green Chem.,2017,19,481–489]。综上,文献报道通过对转酮酶的改造提高了转酮酶对芳香醛的催化活性,其催化芳香醛底物如苯基乙醛,苯基丙醛和苯氧基乙醛产生S立体构型的羟酮产物,而催化苯甲醛的产物构型未知。因此未有文献报道能催化芳香醛尤其是苯甲醛衍生物产生R构型羟酮产物的转酮酶。
高立体选择性的R-芳香二羟酮是酮糖,手性氨基二醇和其他高价值化合物的重要合成砌块,比如R-对甲砜基苯基二羟酮是甲砜霉素和氟苯尼考的手性中间体,R-对硝基苯基二羟酮是氯霉素的手性中间体,而R-苯基羟酮化合物是去甲麻黄碱和去甲伪麻黄碱的手性中间体。因此本发明的R转酮酶突变体能用于手性药物中间体的生物合成,具有一定的应用价值。
因此,本领域的技术人员致力于开发一种高活性和高立体选择性的R转酮酶,使其应用于合成R-芳香二羟酮,提高底物转化率和产物R-芳香二羟酮的光学纯度,满足工业化生产的要求。
发明内容
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是如何获得一种高活性和高立体选择性的R转酮酶,应用于合成R-芳香二羟酮,能提高底物转化率和产物R-芳香二羟酮的光学纯度,满足工业化生产的要求。
为实现上述目的,本发明提供了一种以文献报道的大肠杆菌转酮酶突变体TK/D469T/R520Q/S385Y为母本经过三轮迭代的饱和突变,获取了能高效催化苯甲醛衍生物,包括对甲砜基苯甲醛,对氟苯甲醛,对氯苯甲醛,对溴苯甲醛,对硝基苯甲醛,对甲基苯甲醛和苯甲醛产生R羟酮的转酮酶突变体EcTK1_YYH。
一种高立体选择性R转酮酶突变体,其氨基酸序列为SEQ ID No.1所示;其编码基因的核苷酸序列为SEQ ID No.2所示。
本发明还提供一种高立体选择性R转酮酶突变体的编码基因的重组表达载体。
本发明还提供一种含有高立体选择性R转酮酶突变体的编码基因的重组表达载体的宿主细胞。
进一步地,高立体选择性R转酮酶突变体或编码该突变体的DNA分子或含有编码该突变体的DNA分子的重组表达质粒或含有上述重组表达质粒的宿主细胞均可用于R-芳香二羟酮合成。
本发明还提供一种制备高立体选择性R转酮酶突变体的方法,包括以下步骤:
步骤1、以来源于原核生物大肠杆菌转酮酶的基因作为模板,通过PCR定点突变和饱和突变得到高立体选择性R转酮酶突变体DNA分子;
步骤2、构建含有步骤1得到的高立体选择性R转酮酶突变体DNA分子的重组表达载体;
步骤3、将含有步骤2得到的重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体;
步骤4、通过亲和层析方法,对步骤3获得的具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化,得到了高活性的高立体选择性R转酮酶突变体蛋白。
进一步地,步骤1中DNA分子以来源于原核生物大肠杆菌转酮酶的基因作为模板,通过PCR定点突变和饱和突变得到。
进一步地,步骤1中以重组质粒pET28a-TK为模板。
进一步地,PCR定点突变采用的如D469T-F、D469T-R、R520Q-F、R520Q-R、S385Y-F和S385Y-R所示的双引物对,D469T-F、D469T-R、R520Q-F、R520Q-R、S385Y-F和S385Y-R的核苷酸序列分别如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQID No.9所示。PCR饱和突变采用的是H26-F、H26-R、F434-F、F434-R、L466-F和L466-R所示的双引物对,H26-F、H26-R、F434-F、F434-R、L466-F和L466-R的核苷酸序列分别如ID No.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13、SEQ ID No.14、SEQ ID No.15所示。
进一步地,转酮酶突变体是由原核生物大肠杆菌转酮酶的氨基酸序列通过定点突变和迭代的饱和突变而产生的;原核生物大肠杆菌转酮酶的氨基酸序列第385位的丝氨酸突变为酪氨酸,第469位的天冬氨酸突变为苏氨酸,第520位的精氨酸突变为谷氨酰胺,第26位的组氨酸突变为酪氨酸,第434位的苯丙氨酸突变为酪氨酸,第466位的亮氨酸突变为组氨酸。
进一步地,步骤4获得了高活性和高立体选择性的R转酮酶突变体EcTK1_YYH。
进一步地,结果表明,EcTK1_YYH突变体酶催化对甲砜基苯甲醛合成对甲砜基苯基二羟酮的立体选择性为95.2%ee(R)。
本发明还提供高立体选择性R转酮酶突变体在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
进一步地,以对甲砜基苯甲醛为底物,以高立体选择性R转酮酶突变体为催化酶,将对甲砜基苯甲醛合成R-对甲砜基苯基二羟酮。
本发明还提供高立体选择性R转酮酶突变体的编码基因在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
进一步地,构建含有高立体选择性R转酮酶突变体的编码基因的重组表达载体;含有重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体;通过亲和层析方法,对具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化,得到了高活性的高立体选择性R转酮酶突变体蛋白;以对甲砜基苯甲醛为底物,以高活性的高立体选择性R转酮酶突变体蛋白为催化酶,将对甲砜基苯甲醛合成所述R-对甲砜基苯基二羟酮。
在本发明的较佳实施方式实施例1中,详细说明了对来源于原核生物大肠杆菌的天然转酮酶的定点突变和饱和突变及筛选高活性和高立体选择性的R转酮酶。
在本发明的另一较佳实施方式实施例2中,详细说明了高立体选择性R转酮酶突变体的表达纯化过程。
在本发明的另一较佳实施方式实施例3中,详细说明了高立体选择性R转酮酶突变体EcTK1催化对甲砜基苯甲醛获得高立体选择性产物的检测过程。
本发明通过定点突变和迭代的饱和突变的技术对来源于原核生物大肠杆菌的天然转酮酶的氨基酸序列进行改造,获得了高活性和高立体选择性的R转酮酶突变体EcTK1_YYH,其催化产生的对甲砜基苯基二羟酮的立体选择性为95.2%ee(R)。其有益的技术效果体现为:1.R转酮酶突变体的高活性;2.可以催化芳香醛底物,产生R-芳香二羟酮;3.R-芳香二羟酮的高立体选择性;4.R-芳香二羟酮是重要的手性药物中间体,R转酮酶突变体能用于手性药物中间体的生物合成。本发明获得的突变体为其在手性R-芳香二羟酮合成中的应用奠定了基础。目前,这是转酮酶高效催化芳香醛形成高立体选择性的R-芳香二羟酮的首次研究报道。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例3中,高立体选择性R转酮酶催化对甲砜基苯甲醛的转酮反应示意图;
图2是本发明的一个较佳实施例3,高立体选择性R转酮酶催化对甲砜基苯甲醛获得高立体选择性产物转换成氨醇非对映异构体的反应示意图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
实施例1:定点突变和饱和突变及筛选
对来源于原核生物大肠杆菌的天然转酮酶的定点突变和饱和突变及筛选高活性和高立体选择性的R转酮酶。所使用到的引物核苷酸序列如下表1所示,表1中N代表A、T、C和G任意碱基,K代表T或G碱基,NNK简并密码子可编码随机20种氨基酸。M代表A或C碱基,MNN与NNK互补配对。
表1引物核苷酸序列
(1)以重组质粒pET28a-TK为模板,其核苷酸序列如SEQ ID No.3所示,以D469T-F和D469T-R为引物,利用Phanta Max聚合酶(购自诺唯赞)进行PCR扩增(95℃5min;95℃30s,65℃30s,72℃7.5min,34个循环;72℃10min);PCR产物经FD-Dpn I消化(30℃培养箱,6h)后直接转化至E.coli BL21(DE3)感受态细胞,复苏液充分吹吸混匀,涂布约1/8复苏液于卡那霉素抗性LB平板,37℃培养12-16h。
(2)挑取上述稀释平板上的3个单菌落于卡那霉素抗性LB培养基中37℃培养7-8h,一部分培养液用于测序,另一部分培养液暂时置于4℃冰箱短期保存。测序阳性的转化子培养液以1%的接种量转接至新鲜的含卡那霉素抗性的5mL LB培养基中。培养过夜,抽提质粒,以该质粒(pET28a-TK/D469T)为模板,以R520Q-F和R520Q-R为引物扩增全质粒,重复以上质粒构建方法。继而抽提pET28a-TK/D469T/R520Q质粒,以该质粒为模板,以S385Y-F和S385Y-R为引物扩增全质粒,重复上述方法构建pET28a-TK/D469T/R520Q/S385Y(pET28a-EcTK1)质粒。以pET28a-EcTK1质粒为模板,以H26-F和H26-R为引物扩增全质粒,重复上述方法,获取重组子文库。
(3)挑取上述稀释平板上的所有单菌落(约40个)于卡那霉素抗性LB培养基中37℃培养7-8h,一部分培养液用于测序,另一部分培养液暂时置于4℃冰箱短期保存。测序阳性的转化子培养液以1%的接种量转接至装有新鲜的含卡那霉素抗性的5mL LB培养基的24孔深孔板中,37℃条件下培养3h后,加入0.4mM IPTG诱导剂30℃培养5h,诱导重组基因的高效表达,重组细胞经4000rpm离心收集菌体,-80℃保藏。以同样诱导方法获得pET28a-EcTK1重组菌体作为对照。
(4)将获得的重组菌体加入300ul的50mM Tris-Cl(pH=7.5)悬浮,超声破碎,低温离心,获取上清。在含有5mM对甲砜基苯甲醛和30uM R-转氨酶纯蛋白的反应液中加入20%v/v的上清,30℃反应6h。由于全菌中含有酮醇异构酶,后者会加速二羟酮的消旋,因此为了准确考察转酮产物二羟酮的立体选择性,偶联了一个立体专一性的R-转氨酶,将二羟酮瞬时转化成稳定的手性氨基二醇化合物,通过检测氨基二醇的手性来考察二羟酮的手性。
(5)将(4)所述反应产物采用液相色谱进行检测,考察(1S,2R)-氨基二醇和(1R,2R)-氨基二醇的比例。液相分析条件为:色谱柱super-C18(250mm×4.6mm,粒径5μm),A相:H2O(含10mM KH2PO4,pH=8.5),B相:乙腈,色谱条件:0min 98:2到20min 90:10线性变化,1ml/min,224nm,(1S,2R)-氨基二醇和(1R,2R)-氨基二醇的保留时间分别为9.7和10.9min。结果表明EcTK1_Y在饱和突变体中的R立体选择性最高,并且其活性也较高,因此选用EcTK1_Y为下一轮饱和突变的模板,以F434-F/R为引物进行全质粒PCR,重复上述质粒构建,表达和检测方法。在第二轮饱和突变体中EcTK1_YY的R立体选择性最高,因此将其作为第三轮饱和突变的模板,以L466-F/R为引物,重复上述步骤。最终获得EcTK1_YYH突变体。
实施例2:转酮酶突变体的表达纯化
突变体EcTK1_YYH的表达纯化方法:在LB固体培养基上挑取含重组质粒的大肠杆菌E.coli BL21(DE3)单菌落,接种至40ml LB液体培养基(含50μg/ml卡那霉素抗生素),37℃,220rpm培养过夜。将7.5ml细菌培养液转移至含500ml液体LB培养基的2L摇瓶中,接种两瓶,37℃,220rpm培养至OD600达到0.6-0.8,加入0.4mM IPTG诱导,于30℃,200rpm下诱导培养5h。收集1L发酵液,于5000rpm,离心20min收集细胞,将收集得到的细胞重悬于30mL镍柱结合缓冲液中,并置于冰水混合物中。超声破碎条件:工作5s,停顿10s,总计30min。将经过破碎处理后的混合物,于12,000rpm离心1h后,上清液过0.22μm滤膜过滤。将过滤后的样品上样经镍柱结合缓冲液预先平衡过的2ml镍填料,用10倍柱体积的50mM咪唑洗脱缓冲液冲洗杂蛋白,然后用5ml 250mM咪唑洗脱缓冲液洗脱目的蛋白,分管接样,每管500ul。微量紫外-可见光分光光度计(NanoDrop)测定每管的蛋白浓度,合并浓度较高的几管蛋白液,稀释或者浓缩至2.5ml,上样经甘油缓冲液平衡过的脱盐柱,蛋白液流干后,加入3.5ml甘油缓冲液洗脱蛋白。
实施例3:考察EcTK1突变体催化对甲砜基苯甲醛获得产物的立体选择性
高立体选择性R转酮酶突变体EcTK1催化对甲砜基苯甲醛获得高立体选择性产物的检测过程,所涉及到的化学反应包括转酮反应和转氨反应。
如图1所示,转酮反应的原理是以对甲砜基苯甲醛,为底物,该底物以“1”表示,转酮酶突变体为催化剂,该催化剂以“TK”表示,LiHPA、MgCl2和ThDP存在下,生成S-二羟酮和R-二羟酮产物,S-二羟酮以“2a:S”表示,R-二羟酮产物以“2b:R”表示。
如图2所示,转氨反应的原理是将转酮反应产物二羟酮瞬时转化成稳定的手性氨基二醇化合物,通过检测氨基二醇的立体选择性来考察二羟酮的立体选择性。转酮反应产物S-二羟酮和R-二羟酮,在D-Ala、PLP、NADH、LDH和转氨酶ATA117_AC的作用下,分别转化成(1S,2R)-氨醇和(1R,2R)-氨醇,其中(1S,2R)-氨醇以“3a:(1S,2R)”表示,(1R,2R)-氨醇以“3b:(1R,2R)”表示。液相检测(1S,2R)-氨醇和(1R,2R)-氨醇的浓度,用以分别表征转酮产物2a和2b的浓度。
(1)反应体系共100μl(50mM Tris-Cl buffer,pH 7),5mM对甲砜基苯甲醛,25mMLiHPA,9mM MgCl2,4.8mM ThDP,100μM转酮酶突变体纯蛋白,25℃反应20min,加入18mMEDTA螯合Mg2+终止转酮反应。吸取20μl转酮反应液于新的100μl体系,加入200mM D-Ala,2mMPLP,20mM NADH,90U/ml LDH和200μM转氨酶ATA117_AC于25℃反应30min。S-和R-羟酮产物因此完全转化成(1S,2R)-和(1R,2R)-氨醇非对映异构体。通过检测氨醇非对映异构体的浓度来考察羟酮对映异构体的浓度。
(2)氨醇产物的分析采用安捷伦液相,色谱柱super-C18柱(4.6×250mm,粒径5μm),A相:H2O(含10mM KH2PO4,pH=8.5),B相:乙腈,1ml/min,224nm,(1S,2R)-和(1R,2R)-氨醇的保留时间分别为9.7和10.9min。色谱条件如表2所示:
表2色谱条件
(3)以大肠杆菌EcTK1为母本,三轮迭代的饱和突变中,其关键突变体分别催化5mM对甲砜基苯甲醛底物,25℃20min,所产生R-羟酮的转化率C(%)和立体选择率ee(%)如表3所示。
表3 R-羟酮的转化率C(%)和立体选择率ee(%)
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
序列表
<110> 上海交通大学
<120> 一种高立体选择性R转酮酶突变体及其编码基因和应用
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gacctggcgc cgtataacct gaccctgtgg tctggttcta aagcaatcaa cgaagatgct 1200
gcgggtaact acatccacta cggtgttcgc gagttcggta tgaccgcgat tgctaacggt 1260
atctccctgc acggtggctt cctgccgtac acctccacct atctgatgtt cgtggaatac 1320
gcacgtaacg ccgtacgtat ggctgcgctg atgaaacagc gtcaggtgat ggtttacacc 1380
cacgactcca tcggtcatgg cgaaaccggc ccgactcacc agccggttga gcaggtcgct 1440
tctctgcgcg taaccccgaa catgtctaca tggcgtccgt gtgaccaggt tgaatccgcg 1500
gtcgcgtgga aatacggtgt tgagcgtcag gacggcccga ccgcactgat cctctcccaa 1560
cagaacctgg cgcagcagga acgaactgaa gagcaactgg caaacatcgc gcgcggtggt 1620
tatgtgctga aagactgcgc cggtcagccg gaactgattt tcatcgctac cggttcagaa 1680
gttgaactgg ctgttgctgc ctacgaaaaa ctgactgccg aaggcgtgaa agcgcgcgtg 1740
gtgtccatgc cgtctaccga cgcatttgac aagcaggatg ctgcttaccg tgaatccgta 1800
ctgccgaaag cggttactgc acgcgttgct gtagaagcgg gtattgctga ctactggtac 1860
aagtatgttg gcctgaacgg tgctatcgtc ggtatgacca ccttcggtga atctgctccg 1920
gcagagctgc tgtttgaaga gttcggcttc actgttgata acgttgttgc gaaagcaaaa 1980
gaactgctgt aa 1992
<210> 3
<211> 7288
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atgatgtcct cacgtaaaga gcttgccaat 5160
gctattcgtg cgctgagcat ggacgcagta cagaaagcca aatccggtca cccgggtgcc 5220
cctatgggta tggctgacat tgccgaagtc ctgtggcgtg atttcctgaa acacaacccg 5280
cagaatccgt cctgggctga ccgtgaccgc ttcgtgctgt ccaacggcca cggctccatg 5340
ctgatctaca gcctgctgca cctcaccggt tacgatctgc cgatggaaga actgaaaaac 5400
ttccgtcagc tgcactctaa aactccgggt cacccggaag tgggttacac cgctggtgtg 5460
gaaaccacca ccggtccgct gggtcagggt attgccaacg cagtcggtat ggcgattgca 5520
gaaaaaacgc tggcggcgca gtttaaccgt ccgggccacg acattgtcga ccactacacc 5580
tacgccttca tgggcgacgg ctgcatgatg gaaggcatct cccacgaagt ttgctctctg 5640
gcgggtacgc tgaagctggg taaactgatt gcattctacg atgacaacgg tatttctatc 5700
gatggtcacg ttgaaggctg gttcaccgac gacaccgcaa tgcgtttcga agcttacggc 5760
tggcacgtta ttcgcgacat cgacggtcat gacgcggcat ctatcaaacg cgcagtagaa 5820
gaagcgcgcg cagtgactga caaaccttcc ctgctgatgt gcaaaaccat catcggtttc 5880
ggttccccga acaaagccgg tacccacgac tcccacggtg cgccgctggg cgacgctgaa 5940
attgccctga cccgcgaaca actgggctgg aaatatgcgc cgttcgaaat cccgtctgaa 6000
atctatgctc agtgggatgc gaaagaagca ggccaggcga aagaatccgc atggaacgag 6060
aaattcgctg cttacgcgaa agcttatccg caggaagccg ctgaatttac ccgccgtatg 6120
aaaggcgaaa tgccgtctga cttcgacgct aaagcgaaag agttcatcgc taaactgcag 6180
gctaatccgg cgaaaatcgc cagccgtaaa gcgtctcaga atgctatcga agcgttcggt 6240
ccgctgttgc cggaattcct cggcggttct gctgacctgg cgccgtctaa cctgaccctg 6300
tggtctggtt ctaaagcaat caacgaagat gctgcgggta actacatcca ctacggtgtt 6360
cgcgagttcg gtatgaccgc gattgctaac ggtatctccc tgcacggtgg cttcctgccg 6420
tacacctcca ccttcctgat gttcgtggaa tacgcacgta acgccgtacg tatggctgcg 6480
ctgatgaaac agcgtcaggt gatggtttac acccacgact ccatcggtct gggcgaagac 6540
ggcccgactc accagccggt tgagcaggtc gcttctctgc gcgtaacccc gaacatgtct 6600
acatggcgtc cgtgtgacca ggttgaatcc gcggtcgcgt ggaaatacgg tgttgagcgt 6660
caggacggcc cgaccgcact gatcctctcc cgtcagaacc tggcgcagca ggaacgaact 6720
gaagagcaac tggcaaacat cgcgcgcggt ggttatgtgc tgaaagactg cgccggtcag 6780
ccggaactga ttttcatcgc taccggttca gaagttgaac tggctgttgc tgcctacgaa 6840
aaactgactg ccgaaggcgt gaaagcgcgc gtggtgtcca tgccgtctac cgacgcattt 6900
gacaagcagg atgctgctta ccgtgaatcc gtactgccga aagcggttac tgcacgcgtt 6960
gctgtagaag cgggtattgc tgactactgg tacaagtatg ttggcctgaa cggtgctatc 7020
gtcggtatga ccaccttcgg tgaatctgct ccggcagagc tgctgtttga agagttcggc 7080
ttcactgttg ataacgttgt tgcgaaagca aaagaactgc tgtaactcga gcaccaccac 7140
caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc 7200
accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 7260
ttgctgaaag gaggaactat atccggat 7288
<210> 4
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tccatcggtc tgggcgaaac cggcccgact caccagccgg ttga 44
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggctggtgag tcgggccggt ttcgcccaga ccgatggagt cgtg 44
<210> 6
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
accgcactga tcctctccca acagaacctg gcgcagcagg aacgaa 46
<210> 7
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctgctgcgcc aggttctgtt gggagaggat cagtgcggtc gggccg 46
<210> 8
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tctgctgacc tggcgccgta taacctgacc ctgtggtctg gttcta 46
<210> 9
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agaccacagg gtcaggttat acggcgccag gtcagcagaa ccgccg 46
<210> 10
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 10
aagccaaatc cggtnnkccg ggtgccccta tgggtat 37
<210> 11
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 11
ataggggcac ccggmnnacc ggatttggct ttctgta 37
<210> 12
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 12
cgtacacctc caccnnkctg atgttcgtgg aatacgc 37
<210> 13
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 13
tccacgaaca tcagmnnggt ggaggtgtac ggcagga 37
<210> 14
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 14
acgactccat cggtnnkggc gaaaccggcc cgactca 37
<210> 15
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 15
gggccggttt cgccmnnacc gatggagtcg tgggtgt 37
Claims (10)
1.一种高立体选择性R转酮酶突变体,其特征在于,其氨基酸序列为SEQ ID No.1所示。
2.如权利要求1所述的高立体选择性R转酮酶突变体,其特征在于,其编码基因的核苷酸序列为SEQ ID No.2所示。
3.含有权利要求2所述的高立体选择性R转酮酶突变体的编码基因的重组表达载体。
4.含有权利要求3所述的高立体选择性R转酮酶突变体的编码基因的重组表达载体的宿主细胞。
5.一种制备权利要求1所述高立体选择性R转酮酶突变体的方法,其特征在于,所述方法包括以下步骤:
步骤1、以来源于原核生物大肠杆菌转酮酶的基因作为模板,通过PCR定点突变和饱和突变得到高立体选择性R转酮酶突变体DNA分子;
步骤2、构建含有步骤1得到的高立体选择性R转酮酶突变体DNA分子的重组表达载体;
步骤3、将含有步骤2得到的重组表达载体的宿主细胞通过IPTG诱导的方式大量产生获得具有生物活性的高立体选择性R转酮酶突变体;
步骤4、通过亲和层析方法,对步骤3获得的具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化,得到了高活性的高立体选择性R转酮酶突变体蛋白。
6.如权利要求5所述的一种制备高立体选择性R转酮酶突变体的方法,其特征在于,所述转酮酶突变体是由所述原核生物大肠杆菌转酮酶的氨基酸序列通过所述定点突变和迭代的所述饱和突变而产生的;所述原核生物大肠杆菌转酮酶的氨基酸序列第385位的丝氨酸突变为酪氨酸,第469位的天冬氨酸突变为苏氨酸,第520位的精氨酸突变为谷氨酰胺,第26位的组氨酸突变为酪氨酸,第434位的苯丙氨酸突变为酪氨酸,第466位的亮氨酸突变为组氨酸。
7.权利要求1所述的高立体选择性R转酮酶突变体在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
8.权利要求2所述的高立体选择性R转酮酶突变体的编码基因在催化芳香醛形成高立体选择性的R-芳香二羟酮中的应用。
9.按照权利要求7所述的应用,其特征在于,以对甲砜基苯甲醛为底物,以所述高立体选择性R转酮酶突变体为催化酶,将所述对甲砜基苯甲醛合成R-对甲砜基苯基二羟酮。
10.按照权利要求8所述的应用,其特征在于,构建含有所述高立体选择性R转酮酶突变体的编码基因的重组表达载体;含有所述重组表达载体的宿主细胞通过所述IPTG诱导的方式大量产生获得所述具有生物活性的高立体选择性R转酮酶突变体;通过所述亲和层析方法,对所述具有生物活性的高立体选择性R转酮酶突变体蛋白进行分离纯化,得到所述高活性的高立体选择性R转酮酶突变体蛋白;以所述对甲砜基苯甲醛为底物,以所述高活性的高立体选择性R转酮酶突变体蛋白为催化酶,将所述对甲砜基苯甲醛合成所述R-对甲砜基苯基二羟酮。
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110162619.2A CN114875004B (zh) | 2021-02-05 | 2021-02-05 | 一种高立体选择性r转酮酶突变体及其编码基因和应用 |
CN202280019980.2A CN117157395A (zh) | 2021-02-05 | 2022-01-28 | 一种高立体选择性r转酮酶突变体及其编码基因和应用 |
BR112023015718A BR112023015718A2 (pt) | 2021-02-05 | 2022-01-28 | Mutante de r-cetolase altamente estereosseletivo, vetor de expressão recombinante, célula hospedeira, método para preparar um mutante de r trans-cetonase altamente estereosseletivo, aplicação do mutante e gene que codifica o mutante de r trans-cetolase altamente estereosseletivo e sequência na qual ocorre uma mutação de aminoácido |
KR1020237029610A KR20230137996A (ko) | 2021-02-05 | 2022-01-28 | 고 입체 선택성 r트랜스케톨라제 돌연변이체와 그 코딩 유전자 및 응용 |
EP22749122.2A EP4289947A1 (en) | 2021-02-05 | 2022-01-28 | High-stereoselectivity r transketolase mutant, and encoding gene and use thereof |
MX2023009214A MX2023009214A (es) | 2021-02-05 | 2022-01-28 | Un mutante de las transcetolasas r con alta estereoselectividad y su gen codificante y aplicacion. |
PCT/CN2022/074751 WO2022166843A1 (zh) | 2021-02-05 | 2022-01-28 | 一种高立体选择性r转酮酶突变体及其编码基因和应用 |
CL2023002310A CL2023002310A1 (es) | 2021-02-05 | 2023-08-04 | Un mutante de las transcetolasas r con alta estereoselectividad y su gen codificante y aplicación |
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