CN116463310A - 一种粉防己4’-氧甲基转移酶4’-omt及应用 - Google Patents
一种粉防己4’-氧甲基转移酶4’-omt及应用 Download PDFInfo
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- CN116463310A CN116463310A CN202211411600.8A CN202211411600A CN116463310A CN 116463310 A CN116463310 A CN 116463310A CN 202211411600 A CN202211411600 A CN 202211411600A CN 116463310 A CN116463310 A CN 116463310A
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- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
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Abstract
本发明公开了一种粉防己4’‑氧甲基转移酶4’‑OMT及应用,本发明从药用植物粉防己中分离和鉴定了一条新的4’‑氧甲基转移酶,再利用大肠杆菌对该基因进行异源重组表达蛋白,该药用植物中关键酶基因参与调控苄基异喹啉生物碱的合成,奠定了一定的研究基础。相比于其他植物来源的4’‑氧甲基转移酶,本发明的4’‑氧甲基转移酶St4’‑OMT具有更高的底物特异性,催化效率更高,因此具有一定的应用价值。
Description
技术领域
本发明涉及一种粉防己4’-氧甲基转移酶4’-OMT的克隆表达及应用,并公开了其核苷酸序列、氨基酸序列、其酶学性质,属于生物工程技术领域。
背景技术
苄基异喹啉生物碱具有结构和活性多样性,是天然产物领域研究的热点之一。药理学研究表明苄基异喹啉大多活性优异,如罂粟中得到的罂粟碱有解痉作用;粉防己中的汉防己甲素具有防治肝纤维化及抗埃博拉病毒的活性;莲子心中的莲心碱具有广谱的抗心律失常作用。此外,镇痛药***和可待因、抗菌活性的小糵碱和血根碱、止咳药诺斯卡品等已成为临床一线使用的药物。目前药用苄基异喹啉生物碱的获取方式以植物提取为主,但存在苄基异喹啉生物碱含量低、提取废物污染环境、成本高等问题。
虽然结构复杂多样,但苄基异喹啉生物碱生物合成途径的起始步骤却十分相似。以酪氨酸的代谢产物多巴胺和4-羟基-苯乙醛为底物,经去甲乌药碱合酶、去甲乌药碱6-氧甲基转移酶、乌药碱-氮-甲基转移酶、氮甲基乌药碱3’-羟化酶和4’-氧甲基转移酶等多个酶依次催化合成苄基异喹啉类生物碱的共同中间体——牛心果碱((S)-reticuline)。随后通过异构、偶联、重排、甲基化、去甲基化等反应,形成不同骨架类型的苄基异喹啉类生物碱,如***、可待因、小檗碱、血根碱以及罂粟碱等。
4’-氧甲基转移酶(3′-hydroxy-N-methylcoclaurine 4’-O-methyltransferase,EC2.1.1.116,4’-OMT)可以催化3’羟基N甲基乌药碱形成牛心果碱,是苄基异喹啉生物碱合成过程中的关键限速酶。因此,挖掘催化活性高,底物特异性强的4’-氧甲基转移酶是提高苄基异喹啉生物碱含量的迫切现实需要。
发明内容
本发明目的在于从药用植物粉防己中分离和鉴定了一条新的4’-氧甲基转移酶基因,并将其命名为St4’-OMT,利用大肠杆菌对该基因进行异源重组表达蛋白,并公开了其酶学性质及其应用。
本发明采用的技术方案是:
一种粉防己4’-氧甲基转移酶4’-OMT,具有如SEQ ID NO.2所示的氨基酸序列。
所述的粉防己4’-氧甲基转移酶4’-OMT的编码基因,具有如SEQ ID NO.1所示的核苷酸序列。
一种粉防己4’-氧甲基转移酶重组质粒,所述重组质粒中包括所述的粉防己4’-氧甲基转移酶的编码基因。
进一步地,所述重组质粒中的原核表达载体为pET28a。所述重组质粒pET28a-St4’-OMT的序列如SEQ ID NO.4所示,其中原核表达载体pET28a的序列如SEQ ID NO.3所示。其中,构建重组质粒pET28a-St4’-OMT时,先提取粉防己RNA,并将其反转录获得全基因组cDNA。设计4’-氧甲基转移酶基因St4’-OMT的特异性引物,扩增得到4’-氧甲基转移酶基因St4’-OMT全长编码框序列,将扩增得到的4’-氧甲基转移酶基因St4’-OMT全长编码框序列与双酶切后的pET28a质粒片段进行同源重组连接从而构建得到重组表达质粒pET28a-St4’-OMT。
进一步地,4’-氧甲基转移酶基因St4’-OMT的特异性引物如SEQ ID NO.5、SEQ IDNO.6所示。
一种粉防己4’-氧甲基转移酶重组菌株,所述重组菌株包含所述重组质粒的表达载体。
进一步地,通过如下方法构建:
通过将4’-氧甲基转移酶基因St4’-OMT构建至原核表达载体pET28a中得到重组质粒pET28a-St4’-OMT,将阳性重组质粒pET28a-St4’-OMT转入BL21(DE3)感受态细胞中组成重组菌株。重组菌株在诱导剂IPTG的诱导下表达出可溶性蛋白-4’-氧甲基转移酶St4’-OMT。
一种所述粉防己4’-氧甲基转移酶或所述粉防己4’-氧甲基转移酶重组菌株在苄基异喹啉生物碱催化反应中的应用。
所述应用具体为:催化3’羟基-氮甲基乌药碱反应合成牛心果碱。
进一步地,反应体系中还包含辅因子S-腺苷甲硫氨酸,浓度优选为0.1mmol·L-1。
进一步地,所述催化反应的温度为16~45℃,pH为4.0~10.0。
进一步地,所述催化反应的最适温度为30℃,最适pH为8.0。
进一步地,所述粉防己4’-氧甲基转移酶重组菌株催化3’羟基-氮甲基乌药碱反应合成牛心果碱之前,所述粉防己4’-氧甲基转移酶重组菌株在IPTG溶液条件下进行诱导表达。IPTG溶液的浓度优选为0.4mmol·L-1。
本发明的有益效果是:从药用植物粉防己中鉴定出一条新的4’-氧甲基转移酶St4’-OMT,为进一步解析该药用植物中关键酶基因参与调控苄基异喹啉生物碱的合成奠定了一定的研究基础。相比于其他植物来源的4’-氧甲基转移酶,本发明的4’-氧甲基转移酶St4’-OMT具有更高的底物特异性,催化效率更高,因此具有一定的应用价值。
附图说明
图1:pET28a-St4’-OMT的重组载体谱;
图2:SDS-PAGE分析重组工程菌E.coil BL21 IPTG诱导表达情况结果图:M:Marker;1:含pET28a的菌株胞内可溶表达;2:含pET28a-St4’-OMT菌株沉淀表达;3:含pET28a-St4’-OMT菌株胞内可溶表达;
图3:SDS-PAGE分析重组工程菌E.coil BL21胞内可溶蛋白的纯化情况结果图:M:Marker;1:流穿液;2:Elution buffer第一次洗脱;3:Elution buffer第二次洗脱;4:Elution buffer第三次洗脱;5:Elution buffer第四次洗脱。
图4:A:标准品的HPLC分析图(峰1:3’羟基-氮甲基乌药碱;峰2:牛心果碱。);B:纯化后的pET28a-St4’-OMT蛋白(灭活)催化反应产物的HPLC分析图;C:纯化后pET28a-St4’-OMT蛋白(未灭活)催化反应产物的HPLC分析图;
图5:不同温度对4’-氧甲基转移酶活性的影响结果图;
图6:不同pH对4’-氧甲基转移酶活性的影响,图中,实心方块对应pH 4-6,实心圆对应pH 6-8,实心三角对应pH 8-10;
图7:以3’羟基N甲基乌药碱为底物研究4’-氧甲基转移酶的催化动力学参数拟合结果图;
图8:以不同异喹啉生物碱为底物研究4’-氧甲基转移酶的底物特异性对比图。
具体实施方式
实施例1
本实施例为本发明所述的4’-氧甲基转移酶基因的克隆及大肠杆菌工程菌的构建
粉防己全植株总RNA提取及反转录
1.将粉防己全植株洗净放入液氮速冻,使用已经处理过的研钵对其进行充分研磨,取50-100mg粉末进行RNA提取。利用RNA提取试剂盒和反转录试剂盒得到全植株cDNA备用。
2.大肠杆菌感受态的制备
从LB平板上分别挑取大肠杆菌JM109和BL21(DE3)单菌落,接种于20mL LB培养基中,37℃振荡培养过夜至对数生长中后期。然后将菌液按照1:50(v/v)的比例接种于50mLLB培养基中,37℃震荡培养3h左右至OD600=0.5得到培养菌液。4℃5000r/min离心5min,收集菌体加入5mL冰上预冷的Solution A(感受态试剂盒),轻轻晃动离心管使菌体充分悬浮。4℃5000r/min离心5min,收集菌体加入5mL冰上预冷的SolutionB,轻轻晃动离心管使菌体充分悬浮,按照每管100μL进行分装。制作完成得到JM109和BL21感受态细胞。本感受态细胞可存储于-80℃冰箱用于后续DNA的转化实验。
3.PCR扩增4’-氧甲基转移酶基因
1)引物的设计
根据本实验室粉防己转录组数据库中注释的4’-氧甲基转移酶基因St4’-OMT的序列设计引物,引物直接带有同源臂(下划线标示),具体引物设计为:
F:GCAAATGGGTCGCGGATCCATGGAAGAAGCTATGGATGTCCA(SEQ ID NO.5)
R:CGAGTGCGGCCGCAAGCTTCTAAGGAAAGACCTCAATGACTGATT(SEQ ID NO.6)
2)PCR扩增
扩增反应体系为:模板1μL,上下游引物各1μL,dNTP Mix 4μL,5×primeSTARBuffer 10μL,灭菌的双蒸水32.5μL,primeSTAR DNA聚合酶0.5μL。
PCR反应的条件:98℃30s,56℃30s,72℃2min,35个循环,得到PCR产物最后保存于4℃中。
PCR产物经过胶回收试剂盒回收后送浙江尚亚生物有限公司测序,测序所得到4’-氧甲基转移酶基因St4’-OMT基因序列如SEQ ID NO.1所示。
3)双酶切和连接
利用限制性内切酶BamH I与Hind III对pET28a质粒(序列如SEQ ID NO.3所示)进行双酶切,酶切体系为10×Fast Digest Buffer 5μL,pET28a质粒30μL,限制性内切酶BamHI与Hind III各2.5μL,灭菌蒸馏水10μL。上述反应在37℃反应1h后采用胶回收试剂盒进行胶回收,然后与PCR回收产物进行连接。
连接体系为:MultiS 1μL,5×CE MultiS Buffer 2μL,pET28a胶回收产物1μL,PCR回收产物1μL,灭菌蒸馏水5μL。
上述反应在37℃反应30min后冰浴5min,准备转化。
4)转化
1.取1管前述制备的JM109感受态置于冰上融化。
2.将连接反应液全部转入JM109感受态中,并吹打均匀。
3.冰浴30min后,在42℃水浴中热激90s,然后冰浴2min。
4.加入600μL LB培养基,在37℃条件下培养45min。
5.5000r/min离心2min,弃500μL上清,剩余菌体和培养液吹打均匀,涂布含有卡那霉素抗性的平板。
6.37℃过夜倒置培养。
7.挑选5个单克隆重组质粒进行菌落PCR阳性鉴定,并将阳性克隆送浙江尚亚生物进行测序。测序正确的阳性重组质粒命名为pET28a-St4’-OMT(如SEQ ID NO.4所示)。其图谱如图1所示。
8.将阳性重组质粒转入BL21(DE3)感受态得到重组菌株备用。
实施例2
本实施例为本发明所述的重组菌株的诱导表达
1.取100μL实施例1制得的重组菌株加入20mL LB培养基,加入20μL卡那霉素溶液(终浓度为100μg/ml),以空载菌株(pET28a质粒转入BL21(DE3))作为对照,37℃,220rpm,过夜培养得到种子菌液。
2.取上一步骤种子菌液200μL转接加入含20mL LB培养基的三角瓶,加入20μL卡那霉素溶液(终浓度为100μg/ml),培养至OD600=0.5。
3.每个三角瓶中加入50μL的IPTG溶液(终浓度为0.4mmol·L-1),25℃,220rpm,过夜诱导培养。
4.分别取步骤3中诱导的空质粒菌液和带目的基因的重组质粒菌液1mL,8000rpm离心5min,弃上清,再分别加入1mL磷酸缓冲液(pH=8.0)重悬菌体,超声破碎5min。
5.4℃下,8000rpm离心5min,取上清跑蛋白胶验证,如图2所示,重组蛋白可溶性表达。
6.蛋白纯化及酶活性鉴定
通过上述方法,将剩余的pET28a-St4’-OMT发酵液进行菌体富集破碎得到粗酶液。通过AKTA pure蛋白纯化***进行Ni-柱纯化,用含300mM的高浓度咪唑缓冲液梯度洗脱目的蛋白,如图3所示,将吸附在柱子上的蛋白洗脱下来得到4’-氧甲基转移酶备用。
实施例3
取20mmol·L-1磷酸二氢钾和磷酸氢二钾缓冲液88μL(pH=8.0),加入10μL纯酶(终浓度为10μg·ml-1),30℃预热5min,加入1μL 3’羟基N甲基乌药碱(终浓度为2mM),加入1μLS-腺苷甲硫氨酸(终浓度为0.1mmol·L-1)反应15min,加入50μL甲醇终止反应,通过HPLC检测在波长为280nm处的特征峰。HPLC结果如图4所示,本发明所得到的纯酶St4’-OMT可以催化3’羟基N甲基乌药碱进行甲基化反应。
实施例4-7是对纯化得到的蛋白进行酶学性质研究。其反应体系为100μL反应体系,其中底物母液浓度是10mmol·L-1,辅因子为S-腺苷甲硫氨酸,4’-氧甲基转移酶浓度0.63mg·ml-1,反应15min后加入50μL甲醇终止反应,并进行HPLC定量分析。
实施例4
本实施例为本发明所述的研究4’-氧甲基转移酶酶促反应的最适温度值。以3’羟基N甲基乌药碱为底物,在pH=8.0、辅因子浓度为0.1mmol·L-1的条件下,分别测定不同的温度值(16、20、25、30、40、45、50℃)条件下的牛心果碱的产量。如图5所示,可知4’-氧甲基转移酶酶促反应的最适温度值为30℃。
实施例5
本实施例为本发明所述的研究4’-氧甲基转移酶酶促反应的最适pH值。以3’羟基N甲基乌药碱为底物,在30℃、辅因子浓度为0.1mmol·L-1的条件下,分别测定不同的pH(20mmol·L-1Acetic acid-Sodium acetate,pH 4.0-6.0;20mmol·L-1KH2PO4-K2HPO4,pH6.0-8.0;20mmol·L-1Tris-HCl,pH 8.0-10.0)条件下的牛心果碱的产量。如图6所示,可知4’-氧甲基转移酶酶促反应的最适pH值为8.0。
实施例6
本实施例为本发明所述的以3’羟基N甲基乌药碱为底物研究4’-氧甲基转移酶的催化动力学参数。
在最适反应pH和反应温度下测定该4’-氧甲基转移酶催化不同浓度的3’羟基N甲基乌药碱时的反应速率,反应体系中含0.1mmol·L-1辅因子S-腺苷甲硫氨酸,参照Linewear-Burk双倒数作图法,计算Vmax和Km值,进而算出kcat/Km值,如图7所示,Km=36.1317μM,Vmax=0.01263μM·min-1·mg-1,kcat=0.33S-1,kcat/Km=0.0092μM-1·S-1。
实施例7
本实施例为本发明所述的以3’羟基N甲基乌药碱、去甲乌药碱、乌药碱、N甲基乌药碱和防己乙素为底物研究4’-氧甲基转移酶的底物特异性。如图8所示,结果表明该4’-氧甲基转移酶只对3’羟基N甲基乌药碱的催化,具有优异的底物特异性。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干优化改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (9)
1.一种粉防己4’-氧甲基转移酶4’-OMT,其特征在于,具有如SEQ ID NO.2所示的氨基酸序列。
2.权利要求1所述的粉防己4’-氧甲基转移酶4’-OMT的编码基因,其特征在于,具有如SEQ ID NO.1所示的核苷酸序列。
3.一种粉防己4’-氧甲基转移酶重组质粒,其特征在于,所述重组质粒中包括权利要求2所述的粉防己4’-氧甲基转移酶的编码基因。
4.一种粉防己4’-氧甲基转移酶重组菌株,其特征在于,所述重组菌株包含权利要求3所述重组质粒的表达载体。
5.权利要求1所述粉防己4’-氧甲基转移酶或权利要求4所述粉防己4’-氧甲基转移酶重组菌株在苄基异喹啉生物碱催化反应中的应用。
6.根据权利要求5所述的应用,其特征在于,所述应用具体为:催化3’羟基-氮甲基乌药碱反应合成牛心果碱。
7.根据权利要求6所述的应用,其特征在于,反应体系中还包含辅因子S-腺苷甲硫氨酸。
8.根据权利要求6所述的应用,其特征在于,催化反应的温度为16~45℃,pH为4.0~10.0。
9.根据权利要求6所述的应用,其特征在于,所述粉防己4’-氧甲基转移酶重组菌株催化3’羟基-氮甲基乌药碱反应合成牛心果碱之前,所述粉防己4’-氧甲基转移酶重组菌株在IPTG溶液条件下进行诱导表达。
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