CN116855473A - 一种多聚磷酸激酶突变体及其在制备胞二磷胆碱中的应用 - Google Patents
一种多聚磷酸激酶突变体及其在制备胞二磷胆碱中的应用 Download PDFInfo
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- CN116855473A CN116855473A CN202311039387.7A CN202311039387A CN116855473A CN 116855473 A CN116855473 A CN 116855473A CN 202311039387 A CN202311039387 A CN 202311039387A CN 116855473 A CN116855473 A CN 116855473A
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Abstract
本发明公开了一种多聚磷酸激酶突变体及其在制备胞二磷胆碱中的应用,属于生物技术领域。本发明筛选获得了一种野生型多聚磷酸激酶,可在大肠杆菌中高效表达,通过对其定点突变改造,又获得了活性显著提高的多聚磷酸激酶突变体,能够实现CTP的高效率再生并降低生物催化成本。该突变体酶的最适pH为7.5,最适酶活温度为70℃,对70‑90℃的高温环境均表现出较高的耐受性。且将本发明构建的多聚磷酸激酶突变体用于制备胞二磷胆碱,显著提高了CTP再生的可持续性,可使反应4h胞二磷胆碱产量达144mM,转化率为97%。
Description
技术领域
本发明涉及分子生物学和酶工程领域,具体涉及一种多聚磷酸激酶突变体及其应用。
背景技术
多聚磷酸激酶(Polyphosphate kinase,PPK)可以催化核苷三磷酸NTP(ATP/CTP/GTP/UTP)转化为多聚磷酸(PolyP),且该反应为可逆反应,因此它也可以以PolyP作为磷酸供体催化NDP/NMP转化为NDP/NTP,以实现能量的循环(Green Chem,2021,23,828-837)。根据PPK的结构,PPK主要被分为三类:以PolyP和NDP为底物催化合成NTP的PPK1和PPK2,以PolyP和NMP为底物催化合成NTP的PPK3。
目前关于PPK的研究报道大部分集中以聚磷酸为磷酸供体将AMP转化为ADP或者ATP,而以聚磷酸为磷酸供体将CMP转化为CDP或CTP的研究较少。因此,开发一类新型多聚磷酸激酶,能够利用廉价PolyP原料直接将CMP转化为CDP或CTP具有广泛的应用前景。
胞二磷胆碱(又称为CDP-胆碱或CDPC),分子量488.33,分子式C14H26N4O11P2,作为体内磷脂合成的中间体能促进卵磷脂的生物合成,改善脑循环和代谢,恢复神经组织功能,其作为脑代谢激活剂,具有改善记忆、增加智力、修复脑损伤等作用,在临床上广泛用于急性脑外伤、脑手术后思维障碍、治疗帕金森综合征等,是一种重要的细胞代谢改善药物。
现有胞二磷胆碱的合成方法主要分为化学合成法,微生物转化法和酶催化合成法。其中,化学合成法存在转化率低、环境污染严重等问题,因此不适合用作大规模生产。微生物转化法主要是利用废弃啤酒酵母为生物催化剂,以胞苷酸CMP、葡萄糖和磷酸盐为原料,制造胞二磷胆碱,转化率60-70%,但是存在酵母收集困难,废弃酵母催化活性波动较大,转化率不稳定,转化率低,产物纯化困难等缺陷。酶催化合成法早期是以胞苷三磷酸CTP和磷酸胆碱为原料在磷酰胆碱胞苷转移酶的催化下合成胞二磷胆碱,但是CTP成本较高不利于大规模应用。
近年来,生物学工作者针对酶催化合成法成本高的缺点,开发了以胞苷和磷酸胆碱为原料的酶催化合成法。
中国专利CN112481233A中公开的制备方法,以胞苷和氯化胆碱为底物,在胞苷激酶、胞苷酸激酶、聚磷酸激酶、二磷酸核苷激酶、胆碱激酶和磷酸胆碱胞苷转移酶这六种酶的催化下合成胞二磷胆碱,其工艺路线如下:
此方法的缺陷在于,CMP和磷酸胆碱目前价格都比较便宜,为了制备这两种中间产物,需要额外增加两步反应,用酶种类过多,反应控制复杂,产物浓度较低,仅有27mM,产物中残留ATP和ADP及可能的ATP降解物,纯化难度较大,难以实现规模化生产。
发明内容
发明目的:针对现有技术中所存在的过程复杂、添加酶种类多、不利于反应过程控制的缺陷,本发明提供了一种对CMP有高催化活性的多聚磷酸激酶cPPK的突变体,它能够以PolyP为磷酸供体催化CMP生成CTP,无需添加ATP作为磷酸根循环载体,并将该酶与磷酸胆碱胞苷转移酶liC配合,以CMP和磷酸胆碱为底物进行双酶法合成胞二磷胆碱,其工艺路线如下:
技术方案:为了实现上述发明目的,本发明利用基因工程技术对来自于Candidatus Poribacteria bacterium菌的多聚磷酸激酶,进行改造和筛选,构建以六偏磷酸钠为底物的高性能多聚磷酸激酶突变体,从而有利于实现以PolyP为底物的CTP再生系统的工业化应用。
为此,本发明通过定点突变技术对Candidatus Poribacteria bacterium来源的多聚磷酸激酶进行改造,获得以六偏磷酸钠作为底物的高酶活的多聚磷酸激酶突变体,以便高效地催化六偏磷酸钠上的高能磷酸键转移至CMP上,产生CTP。具体而言,本发明包括如下技术方案。
一种多聚磷酸激酶突变体,其氨基酸序列为SEQ ID NO: 1:
MSQPIKLPLGKKIKLDDFDPDYMAGHDEGRRKMRKKLTEITNEIIELQELLYAESKHALLIVLQAMDAGGKDGTIKHVMEAVNPQGCDVVSFKVPSDEEAAHDFLWRAHKAAPRKGKIVIFNRSHYEDVLVTRVHNFVPKSVWKKRYDQINSFEKILSQSQISILKFFLYISKKEQKKRFKNRLENPDKHWKFSPADLRERAYWDSYMEAFEDVFNNCNTKWAPWHIIPSNDKRCRDLIIAETIAKTLKNLNMKYPEPSVDIASINVNDII (SEQ ID NO:1)。
其为野生型多聚磷酸激酶(cPPK) 的氨基酸序列SEQ ID NO:3第80位的G替换为E、第152位的E替换为S、第182位的D替换为N的突变体。
本发明还提供了编码上述多聚磷酸激酶突变体基因。
优选地,编码上述多聚磷酸激酶突变体SEQ ID NO:2 的基因可以是下述核苷酸序列:
ATGTCACAACCCATAAAACTACCATTAGGAAAGAAGATCAAGTTAGATGATTTCGACCCGGATTATATGGCGGGCCATGATGAGGGCCGTCGTAAGATGCGTAAGAAGTTGACCGAAATCACCAATGAGATCATCGAACTCCAAGAGTTGTTGTACGCCGAGAGCAAGCACGCGCTGCTGATCGTCCTGCAAGCTATGGATGCAGGTGGTAAAGACGGCACTATTAAACACGTGATGGAAGCGGTGAACCCGCAGGGTTGTGATGTCGTGAGCTTTAAAGTTCCGTCCGACGAGGAAGCTGCCCACGACTTCCTGTGGCGTGCGCACAAAGCAGCTCCTCGCAAGGGCAAAATTGTGATTTTCAATCGTAGCCATTATGAAGACGTATTGGTTACCCGTGTTCATAATTTTGTTCCGAAATCTGTTTGGAAAAAGAGATACGACCAGATCAACTCGTTTGAGAAAATCCTGAGCCAAAGCCAGATTAGCATTCTTAAGTTCTTCCTGTACATTTCGAAGAAGGAGCAGAAAAAGCGCTTTAAGAACCGCCTGGAAAACCCAGATAAACATTGGAAATTCTCCCCGGCAGACCTGCGTGAACGTGCGTATTGGGATTCCTATATGGAAGCGTTCGAGGACGTGTTTAACAACTGCAACACCAAATGGGCACCGTGGCACATTATCCCGAGTAATGACAAGCGCTGCCGTGATCTGATTATCGCGGAAACCATCGCCAAAACGCTGAAAAACCTGAATATGAAATACCCGGAGCCGAGCGTTGATATTGCGTCTATTAACGTGAACGACATCATC (SEQ ID NO:2)。
本发明还提供了包含所述基因的表达载体。
本发明还提供了转化了上述表达载体的微生物,该微生物可作为宿主用于表达上述多聚磷酸激酶突变体。
优选地,上述微生物选自大肠杆菌,更优选大肠杆菌BL21 (DE3)。
本发明还提供了上述多聚磷酸激酶突变体或其表达微生物在催化制备胞二磷胆碱中的用途。
在催化制备胞二磷胆碱过程中,以胞苷酸CMP、聚磷酸PolyP和磷酸胆碱为底物,使用磷酸胆碱胞苷转移酶作为生物催化剂,并添加多聚磷酸激酶突变体或者其表达微生物作为CTP再生剂,催化制备胞二磷胆碱。
本发明还提供了催化制备胞二磷胆碱的具体步骤:
(a)向反应器中加入反应器中加入1L含有150mmol/L CMP、155mmol/L氯化磷酸胆碱钾盐、100mmol/L六偏磷酸钠的底物溶液,调节所述溶液的pH至8;
(b)向所述溶液中加入100ml的突变体G80E/E152S/D182N粗酶液和100ml磷酸胆碱胞苷转移酶粗酶液,得到反应体系并搅拌均匀;
(c)连续搅拌所述反应体系,控制体系温度在60℃,保持体系的pH在7.0-8.0之间;
(d)4h反应结束后获得含胞二磷胆碱的粗溶液;
(e)将所述粗溶液过滤,除盐,浓缩,结晶,得到胞二磷胆碱成品。
反应体系中可以添加有150mM氯化镁。
有益效果:
与现有多聚磷酸激酶相比,本发明的对来源于Candidatus Poribacteriabacterium的一种多聚磷酸激酶定点突变改造后获得的突变体具有以PolyP为磷酸供体高效催化胞苷酸CMP合成胞苷三磷酸CTP。且该突变体最适酶活温度为70℃,对70-90℃的高温环境均表现出较高的耐受性。耐热酶具有多种优点,如转化率高、反应速度快、底物粘度低等。因此,利用本发明提供的多聚磷酸激酶突变体在胞二磷胆碱的催化生产中大大减少了酶的使用种类和底物种类,简化了催化工艺过程,并且无需额外添加ATP,降低了纯化难度,减少了反应过程中杂质,从而更具有市场竞争力,并且该生产CTP的方法适用于大规模工业化生产。
附图说明
图1 是野生型多聚磷酸激酶cPPK催化CMP生成CTP反应1h和3h的液相检测图谱比较;
图2 是突变体G80E/E152S/D182N催化CMP生成CTP反应1h和3h的液相检测图谱比较;
图3 为pH对突变体G80E/E152S/D182N的酶活性影响;
图4 为温度对突变体G80E/E152S/D182N的酶活性影响;
图5为多聚磷酸激酶在大肠杆菌BL21(DE3)中的蛋白表达的SDS-PAGE图,泳道说明:M为标准Marker; 1号为野生型多聚磷酸激酶cPPK上清;2号为突变体G80E/E152S/D182的上清;3号为突变体G80E/E152S/D182N经60℃热处理后的上清;4号为突变体G80E/E152S/D182N经70℃热处理后的上清;5号为突变体G80E/E152S/D182N经80℃热处理后的上清;6号为突变体G80E/E152S/D182N经90℃热处理后的上清。
具体实施方式
本发明构建的多聚磷酸激酶突变体 SEQ ID NO:1,是Candidatus Poribacteriabacterium菌来源的野生型多聚磷酸激酶(cPPK)的氨基酸序列SEQ ID NO: 3中个别氨基酸发生替换后的新蛋白质,其中野生型多聚磷酸激酶 SEQ ID NO:3的GenBank序列号为MAT75859.1:
MSQPIKLPLGKKIKLDDFDPDYMAGHDEGRRKMRKKLTEITNEIIELQELLYAESKHALLIVLQAMDAGGKDGTIKHVMGAVNPQGCDVVSFKVPSDEEAAHDFLWRAHKAAPRKGKIVIFNRSHYEDVLVTRVHNFVPKSVWKKRYDQINEFEKILSQSQISILKFFLYISKKEQKKRFKDRLENPDKHWKFSPADLRERAYWDSYMEAFEDVFNNCNTKWAPWHIIPSNDKRCRDLIIAETIAKTLKNLNMKYPEPSVDIASINVNDII(SEQ ID NO:3)。
本发明中氨基酸由单字母或三字母代码表示,具有如下含义:A:Ala(丙氨酸);R:Arg(精氨酸);N:Asn(天冬酰胺);D:Asp(天冬氨酸);C:Cys(半胱氨酸);Q:Gln (谷氨酰胺);E:Glu(谷氨酸);G:Gly(甘氨酸);H:His(组氨酸);I:Ile(异亮氨酸);L:Leu(亮氨酸);K:Lys(赖氨酸);M:Met(甲硫氨酸);F:Phe(苯丙氨酸);P:Pro(脯氨酸);S:Ser(丝氨酸);T:Thr(苏氨酸); W:Trp(色氨酸);Y:Tyr(酪氨酸);V:Val(缬氨酸)。
作为构建多聚磷酸激酶突变体的基础模板,野生型多聚磷酸激酶 SEQ ID NO:3的编码基因是核苷酸序列 SEQ ID NO:4:
ATGTCACAACCCATAAAACTACCATTAGGAAAGAAGATCAAGTTAGATGATTTCGACCCGGATTATATGGCGGGCCATGATGAGGGCCGTCGTAAGATGCGTAAGAAGTTGACCGAAATCACCAATGAGATCATCGAACTCCAAGAGTTGTTGTACGCCGAGAGCAAGCACGCGCTGCTGATCGTCCTGCAAGCTATGGATGCAGGTGGTAAAGACGGCACTATTAAACACGTGATGGGTGCGGTGAACCCGCAGGGTTGTGATGTCGTGAGCTTTAAAGTTCCGTCCGACGAGGAAGCTGCCCACGACTTCCTGTGGCGTGCGCACAAAGCAGCTCCTCGCAAGGGCAAAATTGTGATTTTCAATCGTAGCCATTATGAAGACGTATTGGTTACCCGTGTTCATAATTTTGTTCCGAAATCTGTTTGGAAAAAGAGATACGACCAGATCAACGAGTTTGAGAAAATCCTGAGCCAAAGCCAGATTAGCATTCTTAAGTTCTTCCTGTACATTTCGAAGAAGGAGCAGAAAAAGCGCTTTAAGGACCGCCTGGAAAACCCAGATAAACATTGGAAATTCTCCCCGGCAGACCTGCGTGAACGTGCGTATTGGGATTCCTATATGGAAGCGTTCGAGGACGTGTTTAACAACTGCAACACCAAATGGGCACCGTGGCACATTATCCCGAGTAATGACAAGCGCTGCCGTGATCTGATTATCGCGGAAACCATCGCCAAAACGCTGAAAAACCTGAATATGAAATACCCGGAGCCGAGCGTTGATATTGCGTCTATTAACGTGAACGACATCATC(SEQ ID NO:4)。
为了获得酶性能更高的多聚磷酸激酶,本发明对SEQ ID NO:3的基因序列SEQ IDNO:4进行点突变。通过定点突变技术获得一个氨基酸80位甘氨酸位点、152位谷氨酸位点、182位天冬氨酸位点的突变体氨基酸序列,即本发明中具有氨基酸序列SEQ ID NO:1的突变体。
在本发明中,术语“野生(型)”、“野生酶”、“野生型酶”表示相同的意义,都是指多聚磷酸激酶的野生序列SEQ ID NO:3。为了与突变体相区别和表述方便起见,在本发明中可以将野生型多聚磷酸激酶称为“野生(型)多聚磷酸激酶”或者“野生(型)酶”。
本发明的多聚磷酸激酶突变体的氨基酸数量只有271个,且结构明确,因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。
为了在不同微生物中进行蛋白质SEQ ID NO:1的最佳表达,可以针对特定的微生物比如大肠杆菌进行密码子优化。密码子优化是可用于通过增加感兴趣基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常示出对于编码相同氨基酸的一些密码子之一的特殊偏好性。例如,在生长快速的微生物如大肠杆菌中,优化密码子反映出其各自的基因组tRNA库的组成。因此,在生长快速的微生物中,氨基酸的低频率密码子可以用于相同氨基酸的但高频率的密码子置换。因此,优化的DNA序列的表达在快速生长的微生物中得以改良。在本文中所提供的基因序列SEQ ID NO:2是经过密码子优化的核苷酸序列,但本发明的多聚磷酸激酶突变体SEQ ID NO:l表达基因不限于此。
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
上述转化体宿主可以使任何适合表达多聚磷酸激酶的微生物,包括细菌和真菌。
优选微生物是大肠杆菌、毕赤酵母、酿酒酵母或者枯草芽孢杆菌,优选大肠杆菌,更优选大肠杆菌BL21(DE3)。
本发明的多聚磷酸激酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶,包括纯化酶、粗酶、发酵液、载体固定的酶等;所述菌体的形式包括存活菌体和死亡菌体。
在该反应体系中,主催化剂磷酸胆碱胞苷转移酶也可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶,包括纯化酶、粗酶、发酵液、载体固定的酶等;所述菌体的形式包括存活菌体和死亡菌体。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明并非用于限定本发明的范围。
实施例1:野生型多聚磷酸激酶基因的获得
将来源于Candidatus Poribacteria bacterium的聚磷酸激酶的基因针对大肠杆菌做密码子优化,优化后的DNA序列如SEQ ID NO.4所示,并由南京金斯瑞公司合成并重组到表达载体pET-22b(含酶切位点BamH I、Hind III)上,重组质粒转化至E.coli BL21(DE3),获得重组表达菌E.coli BL21-pET22b-cPPK。
实施例2:定点突变提高cPPK酶活
将Candidatus Poribacteria bacterium菌来源的多聚磷酸激酶cPPK与Genbank数据库中己报道的多聚磷酸激酶的氨基酸序列进行同源性比对分析:同时对cPPK进行结构预测,利用Swiss-Model、Phyre2、Discovery Studio等软件对该酶野生型蛋白进行同源建模,并利用CMP、PolyP双底物分子进行分子对接,从而预测野生型酶的催化位点及底物结合位点,并分析这些位点附近氨基酸残基的作用,从而设计cPPK突变体的氨基酸序列。经上述分析,选择对应于SEQ ID NO.3的第80位的G替换为E、第152位的E替换为S、第182位的D替换为N的突变体。
采用定点突变技术获得单点突变或组合突变体,定点突变的引物采用诺唯赞公司提供的CE Design V1.04设计。突变引物见表1。
表1 突变引物
突变体 | 突变引物的核苷酸序列 | 对应序列号 |
G80E-F | AAACACGTGATGGAAGCGGTGAACCCGCAGGGT | SEQ ID NO :5 |
G80E -R | CGCTTCCATCACGTGTTTAATAGTGCCGTCTTT | SEQ ID NO :6 |
E152S-F | CGACCAGATCAACTCGTTTGAGAAAATCCTGAGCCAAA | SEQ ID NO :7 |
E152S-R | AACGAGTTGATCTGGTCGTATCTCTTTTTCCAA | SEQ ID NO :8 |
D182N-F | GCTTTAAGAACCGCCTGGAAAACCCAGATAAA | SEQ ID NO :9 |
D182N-R | CAGGCGGTTCTTAAAGCGCTTTTTCTGCTCCT | SEQ ID NO :10 |
定点突变以pET-22b-cPPK重组质粒为模板,利用Vazyme 2xphanta master mix进行全质粒扩增,按照表2设置反应体系。
表2 定点突变体系
名称 | 体积(μl) |
Vazyme 2xphanta master mix | 12.5μL |
pET-22b-cPPK载体模板 | 1μL |
引物-F | 1μL |
引物-R | 1μL |
ddH2O | 加至25μL |
PCR程序为:94℃预变性3min,94℃变性30s,66℃退火30s,72℃延伸5min,反应30个循环后,再72℃延伸10min,最后4℃保温。PCR反应结束后,加入1μL DpnI 消化酶,37℃反应2h 消化模板,回收纯化后的PCR产物转化到E. coli BL21(DE3)感受态细胞中。
转化方法:取10μL PCR产物与100μL的诺唯赞商用E. coli BL21(DE3)感受态混匀冰浴20min后,42℃热激90s后迅速拿出,冰浴2min,加入800μL的LB液体培养基,37℃,200rpm,复苏60min,取100ul菌液涂布于含有100μg/mL氨苄抗性的固体LB平板上,37℃恒温箱过夜培养。
次日从平板中挑选重组大肠杆菌 BL21三株,将上述重组菌从平板分别接种到装有5 mL含有相应抗性的液体LB培养基(LB (g/L):蛋白胨 10,氯化钠10,酵母提取物5)的50mL的摇管中,加入相应氨苄抗性,在摇床上37℃恒温培养12 h,转速200 rpm。培养结束后抽提质粒,送南京金斯瑞公司进行测序。最后将测序结果与野生型酶蛋白核苷酸序列进行比对,确定突变是否成功。
按照上述方法先进行单个位点突变得到重组表达菌E.coli BL21 (DE3)-pET22b-G80E,E.coli BL21(DE3)-pET22b-G80E再以进行过一次突变后的重组质粒为模板进行二次、三次突变,最后得到了测序正确的包含cPPK突变体的重组表达菌E.coli BL21(DE3)-pET22b-G80E、E.coli BL21(DE3)-pET22b-G80E/E152S、E.coli BL21(DE3)-pET22b-G80E/E152S/D182N。
实施例3:酶的制备
将上述四种重组表达菌接种到含有氨苄青霉素的5mL LB液体培养基(LB (g/L):蛋白胨10,氯化钠10,酵母提取物5)的50mL的摇管中,在摇床上37℃恒温培养8h,转速200rpm。再将培养菌液按2%接种量接入含有100mL诱导培养基TB中(TB(g/l):酵母粉25,胰蛋白胨15,氯化钠10,葡萄糖2,乳糖3)的500mL摇瓶中,于200rpm,37℃培养2h,待OD600达到0.2左右时转16℃诱导24h离心收集菌体。超声破菌,离心取上清液,上清液即为野生cPPK及多聚磷酸激酶突变体G80E、G80E/E152S、G80E/E152S/D182N粗酶液。粗酶液置于-4℃冰箱保存,可用于后续酶的纯化和生物催化制备CDPC。
实施例4:酶的纯化
为了纯化上述野生型cPPK及其突变体。采用GE公司的AKTA prime层析系统,使用5mL的HisTrap HP镍柱亲和层析。层析柱用pH 8.5,0.5 M NaCl,20 mM咪唑,20 mM磷酸钠缓冲(缓冲液A)预平衡,洗脱缓冲液为pH 8.5,0.5 M NaCl,0.5 M咪唑,20 mM磷酸缓冲(缓冲液B),采用0 %-100 %缓冲B梯度洗脱,总洗脱时间为90min,然后将收集的活性蛋白过Sephacryl S-300 凝胶柱进一步脱盐去除杂蛋白,经紫外检测仪监测,在高数值时收集目的蛋白。纯化后的酶-80保存,用于后续测定蛋白浓度和酶活性。
实施例5:野生型cPPK及突变体酶酶活测定
为了验证多聚磷酸激酶突变体具有以PolyP为磷酸供体高效催化CMP合成CTP的能力,设计反应条件:100ml纯化水中加入120mmol/L CMP、120mmol/L六偏磷酸钠、60mmol/L氯化镁,用30%NaOH调整PH至7.0-8.0,分别加入野生型cPPK和三种突变体纯酶液各2ml,60℃恒温水浴摇床反应3小时,每隔1h取反应液样品,加入浓盐酸调整PH至1.5中止反应,12000rpm离心5min,取上清进行液相检测,计算出已转化的CMP的量,得到4种酶的比酶活。
酶活定义:在60℃,每分钟转化1μmol CMP底物的酶量定义为一个酶活力单位U。以野生酶的酶活力为100%,比较突变体相对酶活。
HPLC测定:色谱柱,COSMOSIL PAQ C18 (250 X 4. 6mm , 5μm),流速1mL/min,波长: 272nm,柱温: 30℃,进样量20ul,流动相:0.6%磷酸溶液,用二乙胺调pH至6.6。
表3酶活力比较
酶 | 酶活力(U/ml) | 相对酶活(100%) |
野生型PpnN | 304 | 100 |
G80E | 477 | 157 |
G80E/E152S | 572 | 188 |
G80E/E152S/D182N | 672 | 221 |
酶活计算结果如表3所示,突变体G80E/E152S/D182N酶活力最高达到672U/ml,相对于野生型cPPK提高了2.2倍。图1-2则为反应1h、3h的液相检测结果的图谱比较:野生型cPPK对CMP的转化率为54%,而突变体G80E/E152S/D182N的转化率达99%,催化效率相较于野生型cPPK有明显提升,证明本发明的多聚磷酸激酶突变体G80E/E152S/D182N具有高效催化CMP合成CTP的活性。
实施例6:pH和温度对突变体G80E/E152S/D182N酶活性的影响
6. 1 测定最适pH值
设置7组与实施例5中相同的反应体系,不再使用30%NaOH调整反应PH至7.0-8.0,而是将纯水替换为不同pH的100mM缓冲溶液pH依次为5.0、6.0、6.5、7.0、7.5、8.0、9.0,1h后结束反应并分别取样检测,计算酶活。以最高的酶活力为 100%,比较相对酶活,绘制酶的pH-相对活力曲线。结果由图3可知,突变体G80E/E152S/D182N酶的最适反应pH值为pH7.5。
6.2测定最适反应温度
设置6组与实施例5中相同的反应体系,仅将水浴摇床反应温度依次改为50℃、60℃、70℃、75℃、80℃、90℃,1h后结束反应并分别取样检测,计算酶活。以最高的酶活力为100%,比较相对酶活,绘制酶的温度-相对活力曲线。以酶活最高的样品作为相对酶活100%,比较相对酶活。由图4可知,突变体G80E/E152S/D182N酶的最适反应温度为70℃。
实施例7:突变体G80E/E152S/D182N酶的热稳定性
为了研究酶的热稳定性,将实施例3中得到的突变体G80E/E152S/D182N粗酶分别取1mL,用EP管平均分装成4份,分别置于60℃、70℃、80℃、90℃水浴条件下保温30min。保温结束后12000rpm离心5min,取上清进行SDS-PAGE电泳。结果如图5,在泳道1-2的29KDa处均有明显的特异性条带,表明野生型cPPK和突变体均可以正确表达;泳道3-6则显示突变体G80E/E152S/D182N粗酶的上清经60-90℃高温处理后,29KDa处有任然一条完整明显的特异性条带,证明该突变体在高温下具有良好的耐热性。利用耐热性,在后续的粗酶催化实验中,可以先用高温加热方式除去大量的杂蛋白,目的蛋白由于耐高温得到了保留,因而达到快速简单纯化粗酶液的效果,减少了杂蛋白产生的副反应。
实施例8:胞二磷胆碱CDPC的制备
8.1 粗酶的制备
将本公司实验室保藏的表达磷酸胆碱胞苷转移酶liC(GenBank: WP_011015739.1)的重组表达菌E.coli BL21(DE3)-pET24a-liC以及重组表达菌E.coli BL21(DE3)-pET22b-G80E/E152S/D182N接种到含有相应抗性的25mL LB液体培养基的100mL的摇瓶中,在摇床上37℃恒温培养10h,转速200 rpm。再将培养菌液按2%接种量接入含有400ml诱导培养基TB的2L带挡板的摇瓶中,两种菌各接2个摇瓶,设置转速250rpm,37℃发酵培养2h后转30℃诱导发酵18h离心收集菌体。超声破菌,离心取上清液,上清液即为磷酸胆碱胞苷转移酶liC及多聚磷酸激酶突变体G80E/E152S/D182N粗酶液。突变体G80E/E152S/D182N粗酶液置于80℃水浴锅中保温30min,然后低温下11000rpm,离心20min去除杂蛋白,得到的上清粗酶液置于-4℃冰箱保存,可用于后续生物催化制备CDPC。
8.2 酶催化制备CDPC
向反应器中加入1L含有150mmol/L CMP、150mM MgCl2、155mmol/L 氯化磷酸胆碱钾盐、100mmol/L 六偏磷酸钠的底物溶液,调节pH至7.5。然后加入催化酶,添加量分别为:100ml的突变体G80E/E152S/D182N粗酶液和100ml磷酸胆碱胞苷转移酶粗酶液,搅拌均匀后,在恒温水浴摇床中进行反应。摇床转速设置为150rpm,反应温度控制在60℃,pH保持在7.0-8.0。反应4小时后,得到含粗品CDPC的溶液(含144mmol/L CDPC),经高效液相色谱检测,转化率达97%。经过滤,纯化,干燥,得到产物CDPC。
Claims (10)
1.一种多聚磷酸激酶突变体,其氨基酸序列为SEQ ID NO:1。
2.编码如权利要求1所述多聚磷酸激酶突变体的基因。
3.如权利要求2所述的基因,其特征在于,核苷酸序列为SEQ ID NO:2。
4.包含如权利要求3所述基因的表达载体。
5.转化了如权利要求4所述表达载体的微生物。
6.如权利要求5所述的微生物,其特征在于,所述微生物是大肠杆菌BL21(DE3)。
7.如权利要求l所述多聚磷酸激酶突变体或者如权利要求6所述微生物在催化制备胞二磷胆碱中的用途。
8.如权利要求7所述的用途,其特征在于,以胞苷酸CMP、聚磷酸PolyP和磷酸胆碱为底物,使用磷酸胆碱胞苷转移酶作为生物催化剂,并添加权利要求1所述多聚磷酸激酶突变体或者如权利要求6所述微生物作为CTP再生剂,催化制备胞二磷胆碱。
9.如权利要求8所述的用途,其特征在于,包括以下步骤:
(a)向反应器中加入包含所述底物的溶液,调节所述溶液的pH至8;
(b)向所述溶液中加入所述酶,得到反应体系并搅拌均匀;
(c)连续搅拌所述反应体系,控制体系温度在60℃,保持体系的pH在7.0-8.0之间;
(d)获得含胞二磷胆碱的粗溶液;
(e)将所述粗溶液过滤,除盐,浓缩,结晶,得到胞二磷胆碱成品。
10.如权利要求9所述的用途,其特征在于,反应体系中添加有氯化镁。
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