CN117467723A - 一种α-D-葡萄糖-1-磷酸胸苷转移酶在dTDP-α-D-葡萄糖合成中的应用 - Google Patents
一种α-D-葡萄糖-1-磷酸胸苷转移酶在dTDP-α-D-葡萄糖合成中的应用 Download PDFInfo
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- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供了一种α‑D‑葡萄糖‑1‑磷酸胸苷转移酶在dTDP‑α‑D‑葡萄糖合成中的应用,属于生物技术领域。本发明提供了一种α‑D‑葡萄糖‑1‑磷酸胸苷转移酶在dTDP‑α‑D‑葡萄糖合成中的应用,合成反应的温度为45~55℃,合成体系的pH值为7.0~9.0。本发明提供的α‑D‑葡萄糖‑1‑磷酸胸苷转移酶来源于海洋细菌噬琼胶假交替单胞菌菌株Hao2018,经重组表达获得α‑D‑葡萄糖‑1‑磷酸胸苷转移酶经酶学性质实验,结果表明α‑D‑葡萄糖‑1‑磷酸胸苷转移酶具有良好的耐热性和耐碱性,使制备的dTDP‑α‑D‑葡萄糖可广泛应用于医学研究、制药、食品等领域。
Description
技术领域
本发明属于生物技术领域,具体涉及一种α-D-葡萄糖-1-磷酸胸苷转移酶在dTDP-α-D-葡萄糖合成中的应用。
背景技术
糖类具有多种生物学活性和药理学作用,在众多疾病治疗中发挥不可或缺的作用。其中,糖型的改变是新型药物研发策略中重要的一环。目前,随机糖基化(glycorandomization)是改变糖型最直接有效的方式之一,然而,该领域只有少量报道,缺少核苷酸糖底物是重要原因。
鼠李糖是众多致病细菌细胞壁结构的重要组成成分之一,缺乏RmlA蛋白的致病细菌会因细胞壁合成受阻而无法生长。因此,RmlA是已被鉴定的、可用于抗结核药物开发的潜在靶标。同时,细菌细胞壁中鼠李糖唯一糖前体供体dTDP-鼠李糖并没有实现商品化,并且dTDP-Rha的生物合成途径比较复杂,化学法合成也相对困难。因此,酶法合成受到广泛关注。细菌中dTDP-Rha的生物合成途径是保守的需要四个酶催化其合成。其中,dTDP-Glc是生物合成dTDP-Rha的中间产物。然而,由于dTDP-Glc价格十分昂贵,这阻碍了dTDP-Rha的生物合成和作为药物靶标的研究进程。
不同微生物来源的α-D-葡萄糖-1-磷酸胸苷转移酶具有各自不同的理化性质及生物学特点。目前已经表征的不同来源的α-D-葡萄糖-1-磷酸胸苷转移酶最适反应温度一般为25℃~37℃,最适反应pH一般为7.0~7.5,缺乏耐热和耐碱的α-D-葡萄糖-1-磷酸胸苷转移酶。
发明内容
有鉴于此,本发明的目的在于提供一种α-D-葡萄糖-1-磷酸胸苷转移酶在dTDP-α-D-葡萄糖合成中的应用,在高温和/或碱性体系下实现dTDP-α-D-葡萄糖的合成。
本发明提供了一种α-D-葡萄糖-1-磷酸胸苷转移酶或表达所述α-D-葡萄糖-1-磷酸胸苷转移酶的重组载体或重组菌株在dTDP-α-D-葡萄糖合成中的应用,所述dTDP-α-D-葡萄糖合成的反应温度为45~55℃,所述dTDP-α-D-葡萄糖合成时,合成体系的pH值为7.0~9.0。
优选的,所述dTDP-α-D-葡萄糖合成的反应温度为50℃,所述dTDP-α-D-葡萄糖合成时,合成体系的pH值为8.0。
优选的,所述dTDP-α-D-葡萄糖合成时,反应体系为每5050μL包括以下含量成分:1MpH7.5的Tris-HCl25μL、体积百分含量50%甘油10μL、10mMdTTP1μL、100mMMgCl22.5μL、10mMDTT5μL、10mMD-葡萄糖-1-磷酸5μL、2U焦磷酸酶20μL50μg/mLα-D-葡萄糖-1-磷酸胸苷转移酶2μL,ddH2O补足至50μL。
优选的,所述α-D-葡萄糖-1-磷酸胸苷转移酶的氨基酸序列如SEQ ID NO:1所示。
优选的,所述α-D-葡萄糖-1-磷酸胸苷转移酶的制备方法,包括以下步骤:
提取噬琼胶假交替单胞菌菌株Hao2018的DNA;
将所述噬琼胶假交替单胞菌菌株Hao2018的DNA为模板进行PCR扩增α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列;
将所述α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列克隆至原核表达载体中,得到的重组表达载体转化至原核表达系统中,经培养和诱导,分离和纯化重组蛋白,得到α-D-葡萄糖-1-磷酸胸苷转移酶。
优选的,用于PCR扩增α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列的引物包括核苷酸序列如SEQ ID NO:2所示的正向引物和核苷酸序列如SEQ ID NO:3所示的反向引物。
优选的,PCR扩增的反应体系:2×PhantaMaxMasterMix(DyePlus)25μL,10μM上下游引物各2μL,DNA模板1μL,用ddH2O补充至总体积为50μL;
PCR扩增的反应程序95℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸1min20s,30个循环,72℃延伸10min,4℃保存。
优选的,所述原核表达载体包括pET-16b载体;
所述α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列在原核表达载体的插入位置为NdeI和BamHI。
优选的,所述诱导用试剂为IPTG;
所述IPTG的终浓度包括0.5mM。
优选的,所述分离和纯化重组蛋白的方法为将诱导后的培养液离心,收集菌体,超声破碎,离心收集上清,过Ni柱纯化。
本发明提供了一种α-D-葡萄糖-1-磷酸胸苷转移酶或表达所述α-D-葡萄糖-1-磷酸胸苷转移酶的重组载体或重组菌株在dTDP-α-D-葡萄糖合成中的应用,所述dTDP-α-D-葡萄糖合成的反应温度为45~55℃,所述dTDP-α-D-葡萄糖合成时,合成体系的pH值为7.0~9.0。本发明提供的α-D-葡萄糖-1-磷酸胸苷转移酶来源于海洋细菌噬琼胶假交替单胞菌(P.agarivorans)菌株Hao2018,经原核表达系统重组表达获得α-D-葡萄糖-1-磷酸胸苷转移酶经酶学性质实验,结果表明本发明制备的α-D-葡萄糖-1-磷酸胸苷转移酶的适宜反应pH值为7.0~9.0,适宜反应温度为45~55℃,具有良好的耐热性和耐碱性,使制备的dTDP-α-D-葡萄糖可广泛应用于医学研究、制药、食品等领域。
附图说明
图1为pET-16b-PaHR在大肠杆菌的阳性克隆验证,其中A为DH5α菌落PCR验证结果;B为DH5α质粒PCR验证结果;C为BL21菌落PCR验证;
图2为重组PaHR酶的纯化过程SDS-PAGE;M为250kDa Prestained ProteinMarker;泳道1为超声破碎后的全细胞蛋白样品;泳道2为破碎液离心后的胞内蛋白样品;泳道3为过Ni柱后的杂蛋白样品;泳道4为纯化后的蛋白样品;
图3为P.agarivorans来源的PaHR蛋白酶的二级结构预测结果;注:蓝色代表螺旋结构,正红色代表延伸结构,玫红色代表卷曲结构;
图4为基于不同物种来源的PaHR氨基酸序列的系统发育关系(A)和多序列比对结果(B);
图5为不同物种来源的PaHR基因多序列比对相似度热图,其中1为Mycobacteriumtuberculosis、2为Mycobacteroides abscessus、3为Pseudomonas aeruginosa、4为Pseudoalteromonas haloplanktis、5为P.agarivorans Hao 2018来源的PaHR基因序列;
图6为噬琼胶假交替单胞菌Hao 2018来源重组PaHR酶的比色法检测原理图;
图7为噬琼胶假交替单胞菌Hao2018来源重组PaHR酶的比色法检测,其中A为空白组;B为阳性组;C和D为测定组;
图8为噬琼胶假交替单胞菌Hao2018来源重组PaHR酶学性质表征,其中A为最适反应温度;B为温度稳定性;C为最适反应pH;D为pH稳定性。
具体实施方式
本发明提供了一种α-D-葡萄糖-1-磷酸胸苷转移酶或表达所述α-D-葡萄糖-1-磷酸胸苷转移酶的重组载体或重组菌株在dTDP-α-D-葡萄糖合成中的应用,所述dTDP-α-D-葡萄糖合成的反应温度为45~55℃,所述dTDP-α-D-葡萄糖合成时,合成体系的pH值为7.0~9.0。
在本发明中,所述α-D-葡萄糖-1-磷酸胸苷转移酶的氨基酸序列优选如SEQ IDNO:1所示。所述α-D-葡萄糖-1-磷酸胸苷转移酶来源于海洋细菌噬琼胶假交替单胞菌(P.agarivorans)菌株Hao2018。所述α-D-葡萄糖-1-磷酸胸苷转移酶的制备方法,优选包括以下步骤:提取噬琼胶假交替单胞菌菌株Hao2018的DNA;将所述噬琼胶假交替单胞菌菌株Hao2018的DNA为模板进行PCR扩增α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列;将所述α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列克隆至原核表达载体中,得到的重组表达载体转化至原核表达系统中,经培养和诱导,分离和纯化重组蛋白,得到α-D-葡萄糖-1-磷酸胸苷转移酶。
在本发明中,用于PCR扩增α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列的引物优选包括核苷酸序列如SEQ ID NO:2(5′-cgccatatgatgtattcgttaaaagccccag-3′)所示的正向引物和核苷酸序列如SEQ ID NO:3(5′-cgcggatccttaaaaaacagtttgatctaaaag-3′)所示的反向引物。PCR扩增的反应体系优选如下:2×PhantaMaxMasterMix(DyePlus)25μL,10μM上下游引物各2μL,DNA模板1μL,用ddH2O补充至总体积为50μL。PCR扩增的反应程序优选如下:95℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸1min20s,30个循环,72℃延伸10min,4℃保存。
本发明对所述原核表达载体的种类没有特殊限制,采用本领域所熟知的原核表达载体即可。所述原核表达载体优选包括pET-16b载体。所述α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列在原核表达载体的插入位置为NdeI和BamHI。本发明对所述克隆的方法没有特殊限制,采用本领域所熟知的克隆方法即可,例如经限制性内切酶酶切目标片段和表达载体后,在连接酶的作用下进行连接,得到重组表达载体。双酶切回收的DNA片段和pET-16b载体的摩尔比优选为10:1。得到重组表达载体后,优选利用热激法转化至原核表达系统中。本发明对所述热激法的操作步骤没有特殊限制,采用本领域所熟知的热激法即可。转化后,优选对转化后的原核表达系统进行验证,所述验证的方法优选为将转化后的原核表达系统涂布于加有100μg/mL氨苄霉素的LB平板,挑取白色菌斑,接种到LB液体培养基,37℃摇床培养12h,对菌液进行测序。
在本发明中,所述培养的方法优选为在37℃、150rpm条件下孵育1h。所述诱导用试剂优选为IPTG。所述IPTG的终浓度优选包括0.5mM。诱导的时机优选为培养至菌液OD为0.6时进行诱导。诱导后在16℃低温诱导发酵16~24h,得到培养液。
在本发明中,所述分离和纯化重组蛋白的方法优选为将诱导后的培养液离心,收集菌体,超声破碎,再次离心收集上清,过Ni柱纯化。所述离心的转速优选为13000rpm。所述离心的时间优选为10min。所述再次离心的转速优选为8000rpm,所述再次离心的时间优选为5min。所述再次离心后,收集上清液用0.22μm滤膜过滤后过Ni柱纯化。所述Ni柱纯化的条件优选为纯化步骤为:首先使用5mL RNase free ddH2O过Ni柱,待液体流至Ni柱上2~3mm时,加入5mL的50mM的咪唑PBS平衡Ni柱,待液体流出后,将使用0.22μm的水系滤膜过滤后蛋白样品加入Ni柱中,待蛋白样品流出后,再次加入5mL的50mM的咪唑PBS,待液体流出后,加入5mL的500mM的咪唑PBS,二次过柱后,收集流出液。依次使用10mL的500mM、50mM的咪唑PBS来清洗Ni柱,Ni柱内保留一半体积的RNase free ddH2O保存。最后将流出液置于激活的透析袋中4℃过夜透析。
在本发明中,所述dTDP-α-D-葡萄糖合成时,反应体系优选为每50μL包括以下含量的组分:1MpH7.5的Tris-HCl25μL、体积百分含量50%甘油10μL、10mMdTTP1μL、100mMMgCl22.5μL、10mMDTT5μL、10mMD-葡萄糖-1-磷酸5μL、2U焦磷酸酶20μL50μg/mLα-D-葡萄糖-1-磷酸胸苷转移酶2μL,ddH2O补足至50μL。
在本发明实施例中,所述dTDP-α-D-葡萄糖合成的最适反应温度优选为50℃,所述dTDP-α-D-葡萄糖合成时,合成体系的最适pH值优选为8.0。可见本发明制备的α-D-葡萄糖-1-磷酸胸苷转移酶的具有良好的耐热性和耐碱性特点,使其在制药、食品和化工等领域中具有应用潜力。
下面结合实施例对本发明提供的一种α-D-葡萄糖-1-磷酸胸苷转移酶在dTDP-α-D-葡萄糖合成中的应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
含有α-D-葡萄糖-1-磷酸胸苷转移酶基因(PaHR)的重组大肠杆菌的制备方法,包括:
获取噬琼胶假交替单胞菌DNA:分离自鲍鱼采苗板的噬琼胶假交替单胞菌菌株Hao2018接种于Zobell2216E培养基,25℃培养进行活化。其中Zobell2216E培养基成分为:蛋白胨5.0g,酵母膏1.0g,海盐35.0g,蒸馏水1000mL,pH值7.6~7.8。
将活化的海洋细菌噬琼胶假交替单胞菌接种于Zobell2216E种子培养基中,25℃,100rpm过夜培养。利用FastPureBacteriaDNAIsolationMiniKit(DC103)提取海洋细菌噬琼胶假交替单胞菌Hao2018的总DNA,具体方法如下:
①样品处理:在EP管中加入过夜培养的海洋细菌噬琼胶假交替单胞菌Hao2018悬液1mL(<1.0×109CFU),置于离心机中室温10000rpm(11,500×g)离心1min,倒弃上层培养液体,加入试剂BufferGA230μL后反复振荡,直至EP管底部菌体完全重悬。加入试剂ProteinaseK20μL,再次充分振荡混匀后加入试剂BufferGB250μL,70℃水浴15min。为排除RNA的残留,在水浴后的消化液中加入4μL的RNaseA,振荡30sec后室温放置10min。静置完成后将全部消化液过柱纯化。
②过柱纯化:在消化液中加入180μL无水乙醇,充分振荡后短暂离心以收集EP管内壁液滴。将全部混合液体使用移液器转移至FastPuregDNAMini ColumnsⅢ吸附柱中,12000rpm(13,400×g)高速离心55sec,倒弃下层滤出液。加入利用无水乙醇配置的试剂BufferPB500μL至吸附柱内,12000rpm(13,400×g)高速离心55sec,倒弃下层滤出液。再加入试剂BufferPW600μL至吸附柱,12000rpm高速离心55sec,倒弃下层滤液,重复该步骤一次。随后空管离心2min后开盖5min挥发残存乙醇。将吸附柱重新置于灭菌后的EP管中,向吸附柱中央滤膜处滴加50μLRNAasefreeddH2O,室温放置2min后12000rpm离心55sec。弃吸附柱后,快速关闭EP管。获得噬琼胶假交替单胞菌DNA,产物置于-20℃保存,避免降解。
PCR扩增条件:2×PhantaMaxMasterMix(DyePlus)25μL,上下游引物(10μM)各2μL,DNA模板1μL,加入ddH2O至50μL;
上游引物PaHR-F5′-cgccatatgatgtattcgttaaaagccccag-3′(SEQ ID NO:2)
下游引物PaHR-R5′-cgcggatccttaaaaaacagtttgatctaaaag-3′(SEQ ID NO:3);
PCR反应条件:95℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸1min20s,30个循环,72℃延伸10min,4℃保存。
将PCR产物进行电泳分离,采用胶回收试剂盒,得到纯化的α-D-葡萄糖-1-磷酸胸苷转移酶基因PCR产物。
构建得到重组表达载体pET-16b-PaHR:将上述PCR产物进行NdeI和BamHI双酶切,酶切条件37℃、3h;酶切体系为:NdeI5μL,BamHI5μL,10×KBuffer10μL,0.1%BSA10μL,α-D-葡萄糖-1-磷酸胸苷转移酶基因DNA片段5μg,ddH2O补足至100μL,将酶切产物电泳,采用胶回收试剂盒得到纯化的酶切DNA片段;
采用质粒抽提试剂盒提取pET-16b质粒,进行NdeI和BamHI双酶切,酶切条件37℃、30min,酶切体系为:NdeI5μL,BamHI5μL,10×KBuffer10μL,质量百分含量0.1%BSA10μL,DNA5μg,ddH2O补足至100μL,采用胶回收试剂盒得到纯化的酶切载体;
将经过双酶切回收的DNA片段和pET-16b载体按照10:1的摩尔比例混合,加入T4DNA连接酶,16℃过夜连接,将10μL连接产物加入100μL大肠杆菌BL21(DE3)感受态细胞,冰上孵育30min,42℃水浴热激60s,后加入900μLLB液体培养基37℃孵育1h,150rpm将菌液离心去掉上清液800μL,将剩余菌液将菌体进行重悬,涂布于加有100μg/mL氨苄霉素的LB平板,挑取白色菌斑,接种到LB液体培养基,37℃摇床培养12h,对菌液进行测序,测序结果显示与PaHR的核苷酸序列(SEQ ID NO:4,ATGTATTCGTTAAAAGCCCCAGTACACAAATCTGCTCGTAAAGGTATTATTTTAGCAGGTGGCTCTGGTACTCGCTTGTACCCACTTACTAAAGTGGTAAGCAAACAATTAATGCCAGTGTACGACAAACCCATGATTTTTTACCCGGTATCAACCTTAATGATGGCGGGTATTACCGAAATACTTATTATATCGACCCCTGCCGAGCTACCACGTTTTAAAGAACTACTAGGCGATGGCAGCGCTTGGGGGATTAGCTTTGAATACGTAGAGCAACCAAGTCCAGATGGCCTAGCACAAGCCTTTTTATTAGCCGAAGACTTTTTACAAGGCCAAAGTGCGGCATTAGTATTGGGCGACAACCTGTTTTATGGCCATGACTTGTCGGTATCGTTACAAAACGCAACAGTGTGCGAATACGGTGCAACGGTATTTGGCTACCACGTAGCTAACCCTAAATCGTACGGAGTAGTTGAGTTTGATGAAAACGGTAAAGCTATTTCAATAGAAGAAAAACCCGACAAACCTAAATCGCACTACGCTGTACCTGGCTTGTACTTTTTTGATAACCGCGTGGTTGAATTTGCAAAAAATGTAAAACCGTCAGAGCGTGGCGAGCTAGAAATTACCGATGTAATAGAGCAATACTTAAATAATAAAGAGCTAAATGTAGAAATAATGGGCCGTGGTACTGCTTGGTTAGATACAGGTACGCTAGATGACCTATTAGATGCCGCTAACTTTATTCGCGCAATAGAAAAACGCCAAGGCTTAAAAATTAATTGCCCAGAAGAAATCGCTTACCGTATGGGCTACATAAATGCAGAAGAGCTTAAAAAGCTGGCTAAGCCGCTTAAAAAGTCGGGTTATGGTAAGTACTTGTTATCGCTTTTAGATCAAACTGTTTTTTAA)一致,并将已整合PaHR基因的pET-16b质粒命名为pET-16b-PaHR。如图1所示,pET-16b-PaHR在大肠杆菌BL21(DE3)的阳性克隆验证,结果表明重组大肠杆菌中表达PaHR基因。
实施例2
α-D-葡萄糖-1-磷酸胸苷转移酶的重组表达方法
将重组大肠杆菌的制备:实施例1制备的重组大肠杆菌接种至LB液体培养基,37℃过夜培养,取菌液,以1%接种量加入到100mL的LB液体培养基中,37℃培养,生长至菌液OD为0.6时,加入终浓度0.5mM的IPTG,继续16℃低温诱导发酵16-24h;将诱导表达的大肠杆菌培养液,13000rpm离心10min,去除上清,收集菌体,在40%功率下超声破碎20min,再次离心5min收集上清液,用0.22μm滤膜过滤后过Ni柱纯化,纯化步骤为:首先使用5mLRNasefreeddH2O过Ni柱,待液体流至Ni柱上2-3mm时,加入5mL的50mM的咪唑PBS平衡Ni柱,待液体流出后,将使用0.22μm的水系滤膜过滤后蛋白样品加入Ni柱中,待蛋白样品流出后,再次加入5mL的50mM的咪唑PBS,待液体流出后,加入5mL的500mM的咪唑PBS,二次过柱后,收集流出液。依次使用10mL的500mM、50mM的咪唑PBS来清洗Ni柱,Ni柱内保留一半体积的RNasefreeddH2O保存。最后将流出液置于激活的透析袋中4℃过夜透析。纯化后的蛋白进行SDS-PAGA电泳,即得重组α-D-葡萄糖-1-磷酸胸苷转移酶(重组PaHR酶)。
如图2所示,重组PaHR酶经过纯化后得到33.5kDa的蛋白,与预期的蛋白序列(SEQID NO:1,MYSLKAPVHKSARKGIILAGGSGTRLYPLTKVV SKQLMPVYDKPMIFYPVSTLMMAGITEILIISTPAELPRFKELLGDGSAWGISFEYVEQPSPDGLAQAFLLAEDFLQGQSAALVLGDNLFYGHDLSVSLQNATVCEYGATVFGYHVANPKSYGVVEFDENGKAISIEEKPDKPKSHYAVPGLYFFDNRVVEFAKNVKPSERGELEITDVIEQYLNNKELNVEIMGRGTAWLDTGTLDDLLDAANFIRAIEKRQGLKINCPEEIAYRMGYINAEELKKLAKPLKKSGYGKYLLSLLDQTVF)的预期分子量大小一致,说明本发明成功表达了重组α-D-葡萄糖-1-磷酸胸苷转移酶。
实施例3
噬琼胶假交替单胞菌菌株Hao2018来源的α-D-葡萄糖-1-磷酸胸苷转移酶基因(PaHR)及其蛋白的系统进化分析
PaHR基因的核酸序列含有912个碱基对,共编码303个氨基酸残基,理论分子量(Molecularweight,MW)为33.61kDa,理论等电点(Theoretical pI)为5.42。负电荷残基(Asp+Glu)为38个,正电荷残基为(Arg+Lys)为32个。不稳定系数(Instabilityindex,II)为37.24,表明PaHR酶蛋白是稳定的。PaHR酶蛋白脂肪族氨基酸指数(Aliphaticindex,AI)为95.28,表明PaHR具有良好的热稳定性。同时,PaHR酶蛋白的疏水性指数(Grandaverageofhydropathicity,GRAVY)为-0.114,表明该酶蛋白属于亲水性蛋白。
此外,PaHR酶蛋白在体外培养的mammalianreticulocytes中预测半衰期为30h,在yeast中超过20h,在Escherichiacoli中超过10h。二级结构预测结果显示PaHR酶蛋白含有α螺旋(Alphahelix)33.66%,延伸主链(Extended strand)22.11%和无规则卷曲(Randomcoil)44.22%,无其他二级结构(图3)。
将Hao2018来源的PaHR的氨基酸序列与公布于NCBI的15个不同物种的PaHR酶的氨基酸序列进行多序列比对并构建系统发生树(图4)。这15个物种分别为Escherichiacoli,Klebsiellapneumoniae,Mycobacterium tuberculosis,Streptococcuspneumoniae,Pseudomonasaeruginosa,Listeria monocytogenes,Neisseriameningitidis,Mycobacteroidesabscessus,Vibrio parahaemolyticus,Enterococcusfaecalis,Enterobacterhormaechei,Shigella boydii,Pseudoalteromonashaloplanktis,Pseudoalteromonascarrageenovora,Salmonellaenterica。
比对结果显示参与分析的物种形成了3个独立分支,其中3株Pseudoalteromonas菌株与Mycobacteroidesabscessus和Mycobacterium tuberculosis形成了相对独立的分支(图4中A)。
进一步选取了距离该分支最近的Pseudomonasaeruginosa共6个物种的PaHR进行多序列比对。序列比对相似度如图(图5)。菌株Hao2018来源的PaHR与除Pseudoalteromonas菌属以外的其他菌株相似程度区间为57.99%~64.85%。
实施例3
重组α-D-葡萄糖-1-磷酸胸苷转移酶活性检测
PaHR酶促反应及终止显色均在EP管中进行,待反应完成后,利用酶标仪读取50μL反应产物于630nm的吸光值。具体操作为:首先按表1配制酶促反应体系,于37℃反应10min后,通过添加50μL孔雀石绿显色液(0.35‰(w/v)孔雀石绿、0.5‰(v/v)TritonX-100、2.5‰(w/v)钼酸铵,其中以0.7mol/L的盐酸为溶剂)来终止反应,加入显色液后于37℃显色反应10min后即可读数(图6)。其中,以体系中含有焦磷酸且不含PaHR酶促反应体系为阳性对照组;以不含PaHR酶促反应体系为空白对照组。
表1PaHR酶促反应体系
如图7所示,噬琼胶假交替单胞菌Hao2018来源重组PaHR酶的比色法检测。(A)空白组;(B)阳性组;(C)(D)测定组。
结果表明,PaHR酶具有胸苷转移酶活性,该方法可用于PaHR酶活性定性检测。
实施例4
α-D-葡萄糖-1-磷酸胸苷转移酶学性质分析
(1)最适温度:参照实施例3的酶活性测定方法,将反应体系分别于10、20、30、40、50、60、70、80℃反应10min后,于37℃中终止显色10min测定吸光值。
(2)温度稳定性:将纯化后的PaHR酶液分别于10、20、30、40、50、60℃下水浴1h,然后参照实施例3的酶活性测定方法。
(3)最适pH:参照实施例3的酶活性测定方法,利用缓冲液将反应体系的pH分别调整为4.0、5.0、6.0、7.0、8.0、9.0、10.0反应10min后,于37℃中终止显色10min测定吸光值。
(4)pH稳定性:将纯化后的PaHR酶液分别于pH为5、6、7、8、9、10的条件下水浴1h,然后参照实施例3的酶活性测定方法。
在不同温度和pH下反应10min后,使用比色法测定重组PaHR酶的最适反应温度和pH。如图8所示,在其他反应因素不变的情况下,纯化后的重组PaHR酶在50℃表现出最大活性,故海洋细菌噬琼胶假交替单胞菌来源的重组PaHR酶最适反应温度为50℃,且在50℃的孵育条件下仍保持了较好的热稳定性。同样的,重组PaHR酶在pH为8时具有最优活性,且在碱性条件下具有较好的酶活性以及稳定性。
对比例1
文献《Novel dTDP-L-Rhamnose Synthetic Enzymes(RmlABCD)FromSaccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis ofdTDP-L-Rhamnose》中,ShidaYang等,Frontiers in Microbiology,2021.11.8中公开了制备丁香糖丝菌的α-D-葡萄糖-1-磷酸胸苷转移酶,经序列比对,与α-D-葡萄糖-1-磷酸胸苷转移酶氨基酸序列(SEQ ID NO:1)相似性为57%,采用实施例3记载的比色法检测酶活,结果表明,丁香糖丝菌为来源的α-D-葡萄糖-1-磷酸胸苷转移酶最适温度为37℃,而噬琼胶假交替单胞菌菌株Hao2018来源的α-D-葡萄糖-1-磷酸胸苷转移酶的适宜反应温度为50~55℃。
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来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种α-D-葡萄糖-1-磷酸胸苷转移酶或表达所述α-D-葡萄糖-1-磷酸胸苷转移酶的重组载体或重组菌株在dTDP-α-D-葡萄糖合成中的应用,所述dTDP-α-D-葡萄糖合成的反应温度为45~55℃,所述dTDP-α-D-葡萄糖合成时,合成体系的pH值为7.0~9.0。
2.根据权利要求1所述应用,其特征在于,所述dTDP-α-D-葡萄糖合成的反应温度为50℃,所述dTDP-α-D-葡萄糖合成时,合成体系的pH值为8.0。
3.根据权利要求1所述应用,其特征在于,所述dTDP-α-D-葡萄糖合成时,反应体系中每50μL包括以下含量成分:1MpH7.5的Tris-HCl2.5μL、体积百分含量50%甘油10μL、10mMdTTP1μL、100mMMgCl22.5μL、10mMDTT5μL、10mMD-葡萄糖-1-磷酸5μL、2U焦磷酸酶20μL、50μg/mLα-D-葡萄糖-1-磷酸胸苷转移酶2μL,ddH2O补足至50μL。
4.根据权利要求1所述应用,其特征在于,所述α-D-葡萄糖-1-磷酸胸苷转移酶的氨基酸序列如SEQ ID NO:1所示。
5.根据权利要求1所述应用,其特征在于,所述α-D-葡萄糖-1-磷酸胸苷转移酶的制备方法,包括以下步骤:
提取噬琼胶假交替单胞菌菌株Hao2018的DNA;
将所述噬琼胶假交替单胞菌菌株Hao2018的DNA为模板进行PCR扩增α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列;
将所述α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列克隆至原核表达载体中,得到的重组表达载体转化至原核表达系统中,经培养和诱导,分离和纯化重组蛋白,得到α-D-葡萄糖-1-磷酸胸苷转移酶。
6.根据权利要求5所述应用,其特征在于,用于PCR扩增α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列的引物包括核苷酸序列如SEQ ID NO:2所示的正向引物和核苷酸序列如SEQ IDNO:3所示的反向引物。
7.根据权利要求5所述应用,其特征在于,PCR扩增的反应体系:2×PhantaMaxMasterMix(DyePlus)25μL,10μM上下游引物各2μL,DNA模板1μL,用ddH2O补充至总体积为50μL;
PCR扩增的反应程序95℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸1min20s,30个循环,72℃延伸10min,4℃保存。
8.根据权利要求5所述应用,其特征在于,所述原核表达载体包括pET-16b载体;
所述α-D-葡萄糖-1-磷酸胸苷转移酶的DNA序列在原核表达载体的插入位置为NdeI和BamHI。
9.根据权利要求5所述应用,其特征在于,所述诱导用试剂为IPTG;
所述IPTG的终浓度包括0.5mM。
10.根据权利要求5~9中任意一项所述应用,其特征在于,所述分离和纯化重组蛋白的方法为将诱导后的培养液离心,收集菌体,超声破碎,离心收集上清,过Ni柱纯化。
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