CN114606213B - 一种多聚磷酸激酶突变体、工程菌及其应用 - Google Patents
一种多聚磷酸激酶突变体、工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种多聚磷酸激酶突变体、工程菌及其应用，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列第79位、第106位、第108位、第111位或第285位进行单突变或多突变获得的。本发明提供了多种来源于Cytophaga hutchinsonii的多聚磷酸激酶突变体，这些突变体的比酶活较母本多聚磷酸激酶提升2.7‑17.9倍，这些突变体构成的ATP再生体系能减少70％以上ATP依赖的生物催化合成反应中ATP的消耗，具有广泛的工业应用前景。
Description
(一)技术领域
本发明属于生物工程技术领域，特别涉及一种多聚磷酸激酶(PPK)突变体、工程菌及其应用，开发高效廉价的ATP再生体系。
(二)背景技术
腺嘌呤核苷三磷酸(ATP)是生物体中调节能量代谢、RNA和DNA合成以及信号转导等多种生物过程的关键分子。大量潜在的生物催化剂的活性，包括连接酶、激酶和合成酶，也依赖ATP。在这些生物转化过程中导入ATP再生系统可以显著降低ATP的消耗。大多数ATP再生系统包括磷酸供体以及催化ADP和磷酸供体之间的磷酸转移酶。然而很多有价值的产品的生物合成需要由AMP直接生成ATP的再生系统，如土四环素、1,6-六甲二胺和β-卡碱酰胺等。为了从AMP再生ATP，可以采用将AMP合成ADP的再生系统导入到从ADP开始生成ATP的再生系统。
值得注意的是，多种酶和磷酸供体的使用会使原本的生物转化过程更加复杂。考虑到这些限制，人们对聚磷酸激酶2-III类(PPK2-III)酶进行了深入研究，因为它们可以将AMP转化为ATP。磷酸供体的可及性和稳定性对ATP再生系统应用至关重要。对于PPK2-III酶构成的ATP再生系统，无机聚磷酸盐(polyP)是一种稳定的磷酸供体。在很多研究中，长链polyP被作为磷酸供体。但是，使用短链polyP作为磷酸供体可以使ATP生成系统更具吸引力，因为短链polyP比长链polyP更廉价。最容易获得的polyP是的聚磷酸(PPA)，它是含有不同长度线性多聚磷酸的混合物，大量用于颜料、石油催化剂、香料和阻火剂的制造。在此，我们旨在开发一种基于PPK2-III酶并能以AMP和PPA为底物的的高效廉价ATP再生系统。我们对来自Cytophaga hutchinsonii的多聚磷酸酶(ChPPK)进行了分子改造，以提高其酶学性质，用于构建廉价高效的ATP再生体系。
(三)发明内容
本发明目的是针对现有多聚磷酸激酶活性不高的问题，提供一种多聚磷酸激酶突变体及利用该多聚磷酸激酶突变体基因重组菌或其粗酶液作为生物催化剂，用于ATP再生系统的构建，以解决现有ATP再生系统效率较低的问题。
本发明采用的技术方案是：
本发明提供一种多聚磷酸激酶突变体，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列第79位、第106位、第108位、第111位或第285位进行单突变或多突变获得的；所述SEQ ID NO：2所示氨基酸序列编码基因的核苷酸序列如SEQ ID NO：1所示。
优选的，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列突变为下列之一：(1)第79位丙氨酸突变为甘氨酸(A79G)；(2)第106位丝氨酸突变为半胱氨酸(S106C)；(3)第108位异亮氨酸突变为苯丙氨酸、天冬酰胺或酪氨酸(I108F、I108N、I108Y)；(4)第111位丝氨酸突变为谷氨酸或赖氨酸或丙氨酸(S111E、S111K、S111A)；(5)第285位亮氨酸突变为脯氨酸(L285P)；(6)第79位丙氨酸突变为甘氨酸、第108位异亮氨酸突变为苯丙氨酸(A79G/I108F)；(7)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸(A79G/S106C/I108F)；(8)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第111位丝氨酸突变为丙氨酸(A79G/S106C/I108F/S111A)；(9)第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第285位亮氨酸突变为脯氨酸(A79G/S106C/I108F/L285P)。
本发明还提供一种所述多聚磷酸激酶突变体的编码基因、编码基因构建的重组载体以及重组载体转化宿主菌制备的重组基因工程菌；所述载体为pET表达载体，pCW表达载体或pPIC表达载体，优选质粒pET-28a(+)；所述的宿主细胞为大肠杆菌、枯草芽孢杆菌、链霉菌、酿酒酵母、毕赤酵母或哺乳动物细胞，优选大肠杆菌Escherichia coli(E.coli)BL21(DE3)。
本发明还提供一种所述多聚磷酸激酶突变体在构建ATP再生体系中的应用，所述应用是用多聚磷酸激酶突变体和聚磷酸(PPA)替换部分ATP，所述多聚磷酸激酶突变体以多聚磷酸激酶突变体基因工程菌经发酵培养获得的湿菌体提取的粗酶液或纯酶形式作用。本发明所述多聚磷酸激酶突变体能够替换所有ATP依赖的生物转化反应中部分ATP。
本发明还提供一种所述多聚磷酸激酶突变体在合成烟酰胺单核苷酸(NMN)中的应用，所述应用的方法为：以所述多聚磷酸激酶突变体基因工程菌和烟酰胺核糖激酶(NRK)基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸(ATP)和烟酰胺核糖(NR)为底物，加入氯化镁和聚磷酸(PPA)，以pH6.5的缓冲液为反应介质，在37℃进行反应(优选反应6h)，获得烟酰胺单核苷酸(NMN)。
所述腺嘌呤核苷三磷酸(ATP)加入量以缓冲液体积计为10-100mM，优选25mM；所述NR加入量以缓冲液体积计为50-200mM，优选100mM；所述氯化镁加入量以缓冲液体积计为5-20mM，优选10mM；所述PPA加入量以缓冲液体积计为1-10g/L，优选4.8g/L；所述多聚磷酸激酶突变体上清液加入量以破碎前湿菌体量计为2-30mg/mL缓冲液，优选4mg/mL；所述烟酰胺核糖激酶上清液加入量以破碎前湿菌体量计为5-30mg/mL缓冲液，优选8mg/mL。
优选的，所述缓冲液为pH 6.5、50mM磷酸钾缓冲液。
优选的，所述催化剂按如下方法制备：将多聚磷酸激酶突变体基因工程菌接种至含有50μg/mL卡那霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以1％(v/v)接种量接种至新鲜的含有50μg/mL卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD600达0.6，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、8000rpm离心10min，弃去上清液，收集湿菌体沉淀；收集的湿菌体(优选按40g/L的量)用pH为7.2的50mM磷酸钾缓冲液(PBS)重悬，并使用超声波细胞粉碎机破碎，破碎功率为50W，每工作1s，间歇2s，总共破碎20min；收集细胞裂解液，在12000g下离心1min，取上清液，即为粗酶液。
优选的，所述烟酰胺核糖激酶(NRK)基因工程菌的上清液制备方法同多聚磷酸激酶突变体基因工程菌的上清液。所述NRK氨基酸序列如SEQ ID NO：4所示，核苷酸序列如SEQID NO：3所示，构建工程菌的载体为pET-28a(+)，宿主菌为E.coli BL21(DE3)。
本发明还提供一种所述多聚磷酸激酶突变体在合成葡萄糖6磷酸(G6P)中的应用，所述应用的方法为：以所述多聚磷酸激酶突变体基因工程菌和己糖激酶(HK)基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸(ATP)和葡萄糖为底物，加入氯化镁和聚磷酸(PPA)，以pH 7.2的50mM磷酸钾缓冲液为反应介质，在37℃进行反应(优选8h)，获得G6P。
所述腺嘌呤核苷三磷酸(ATP)加入量以缓冲液体积计为10-100mM，优选25mM；所述葡萄糖加入量以缓冲液体积计为20-150mM，优选100mM；所述氯化镁加入量以缓冲液体积计为5-20mM，优选10mM；所述PPA加入量以缓冲液体积计为1-10g/L，优选4.8g/L；所述多聚磷酸激酶突变体上清液加入量以破碎前湿菌体量计为2-30mg/mL缓冲液，优选4mg/mL；所述HK上清液加入量以破碎前湿菌体量计为5-30mg/mL缓冲液，优选12mg/mL。
优选的，所述缓冲液为pH 7.2、50mM磷酸钾缓冲液。
优选的，所述己糖激酶(HK)基因工程菌上清液的制备同多聚磷酸激酶突变体基因工程菌的上清液。所述HK氨基酸序列如SEQ ID NO：6所示，核苷酸序列如SEQ ID NO：5所示，构建工程菌的载体为pET-28a(+)，宿主菌为E.coli BL21(DE3)。
与现有技术相比，本发明有益效果主要体现在：
本发明提供了多种来源于Cytophaga hutchinsonii的多聚磷酸激酶突变体，这些突变体的比酶活较母本多聚磷酸激酶提升2.7-17.9倍，这些突变体构成的ATP再生体系在合成NMN与G6P的反应中能减少70％以上ATP的使用量而不影响最终产率。因此，本发明具有广泛的工业应用前景。
(四)附图说明
图1为实施例4方法制备的含突变体的粗酶液相对酶活。
图2为实施例5方法制备的突变体相对酶活。
图3为实施例6方法制备的突变体相对酶活。
图4为实施例7中温度对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图5为实施例7中pH对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图6为实施例7中AMP浓度对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图7为实施例7中PPA浓度对突变体ChPPK/A79G/S106C/I108F/L285P和野生型酶活的影响。
图8为实施例9中不同反应体系中NMN产量曲线图。
图9为实施例10中不同反应体系中G6P产量柱状图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例中，未注明具体实验条件的实验方法，通常按照常规条件，如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件进行。
LB平板组成：10g/L胰蛋白胨、10g/L氯化钠、5g/L酵母提取物、15g/L琼脂，溶剂为水，pH自然。
LB液体培养基组成：10g/L胰蛋白胨、10g/L氯化钠、5g/L酵母提取物，溶剂为水，pH自然。
实施例1：野生型Ecoli.BL21(DE3)-ChPPK的构建
根据GenBank中来自哈氏嗜纤维菌(Cytophaga hutchinsonii)的PPK蛋白序列(ChPPK，GenBank号：ABG57400.1)，针对大肠杆菌密码子偏好性优化，并在序列C端融合一个6His标签，由北京擎科生物公司(中国北京)合成长度为930bp的重组基因ChPPK序列，其核苷酸序列如SEQ ID NO：1所示，编码蛋白的氨基酸序列为SEQ ID NO：2所示。
将重组基因ChPPK插入到pET-28a(+)的T7启动子下，得到表达质粒pET28-ChPPK。将该表达质粒转化至大肠杆菌E.coli BL21(DE3)中，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下培养8-12h，挑取阳性克隆，即为野生型Ecoli.BL21(DE3)-ChPPK，用于表达重组ChPPK。
SEQ ID NO：1
ATGGCAACCGATTTTAGCAAACTGAGCAAATATGTTGAAACGCTGCGTGTGAAACCGAAACAGAGCATTGATCTGAAAAAGGATTTTGATACCGATTATGATCATAAAATGCTGACGAAAGAAGAAGGTGAAGAACTGCTGAATCTGGGTATTAGTAAACTGAGCGAAATTCAGGAAAAACTGTATGCATCTGGCACAAAAAGCGTGCTGATTGTTTTTCAGGCAATGGATGCAGCAGGTAAAGATGGTACCGTTAAACATATTATGACGGGTCTGAATCCGCAGGGTGTTAAAGTGACCAGCTTTAAAGTTCCGTCCAAAATTGAACTGAGTCATGATTATCTGTGGCGTCATTATGTGGCACTGCCGGCAACCGGCGAAATTGGTATTTTTAACCGTAGCCATTATGAAAATGTGCTGGTTACCCGTGTACATCCGGAATATCTGCTGAGCGAACAGACCAGCGGTGTTACCGCAATTGAACAGGTAAATCAGAAATTTTGGGATAAACGCTTTCAGCAGATCAATAACTTTGAACAGCATATTAGCGAAAACGGTACCATTGTTCTGAAATTTTTTCTGCATGTTTCCAAAAAGGAACAGAAAAAGCGTTTTATTGAACGTATCGAACTGGATACCAAAAATTGGAAATTTTCAACCGGTGATCTGAAAGAACGTGCCCATTGGAAAGATTATCGTAATGCGTATGAAGATATGCTGGCAAATACCTCTACCAAACAGGCCCCGTGGTTTGTTATTCCGGCCGATGATAAATGGTTTACCCGTCTGCTGATTGCAGAAATTATCTGTACCGAACTGGAAAAACTGAATCTGACCTTTCCGACCGTGAGCCTGGAACAGAAAGCGGAACTGGAAAAAGCAAAAGCAGAACTGGTTGCAGAAAAATCAAGCGATCATCATCATCACCACTAA。
SEQ ID NO：2
MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVTRVHPEYLLSEQTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSLEQKAELEKAKAELVAEKSSDHHHHHH。
实施例2：野生型Ecoli.BL21(DE3)-ChPPK的诱导表达及野生型多聚磷酸激酶的提取
(1)粗酶液：将实施例1获得的野生型Ecoli.BL21(DE3)-ChPPK接种至含有50μg/mL卡那霉素抗性的LB液体培养基，37℃，200rpm下培养12h，再以1％(v/v)接种量接种至新鲜的含有50μg/mL卡那霉素抗性的LB液体培养基中，于37℃，150rpm下培养至菌体OD600＝0.6，加入终浓度为0.1mM的IPTG，28℃下诱导培养12h后，4℃、8000rpm离心10min，弃去上清液，收集沉淀，即获得含有表达重组ChPPK的湿菌体。收集的湿菌体按40g/L的量用pH为7.2的50mM磷酸钾缓冲液(PBS)重悬成菌悬液，并使用超声波细胞粉碎机破碎，破碎功率为50W，每工作1s，间歇2s，总共破碎20min。收集细胞破碎液，在12000rpm下离心1min，取上清，即为粗酶液，后续粗酶液的用量以对应破碎前菌悬液中菌体量计。
(2)纯酶：取5mL粗酶液稀释到40mL磷酸钾缓冲液(20mM，pH 7.2)中，然后上样到GEHealthcare公司的HisTrap HP纯化柱中(10mL柱体积，预先用含500mM氯化钠的pH 7.2、20mM磷酸钾缓冲液冲洗)。上样后的纯化柱用100mL清洗缓冲液(500mM氯化钠+50mM咪唑的pH7.2、20mM磷酸钾缓冲液)以0.5mL/min的速度洗脱，除去结合在纯化柱上的杂蛋白。然后用洗脱缓冲液(500mM氯化钠+250mM咪唑的pH 7.2、20mM磷酸钾缓冲液)以0.5mL/min的速度洗脱，收集含目的蛋白的洗脱液，再用pH7.2的20mM磷酸钾缓冲液在透析袋(截留分子量为14KDa)中透析48h，取截留液，即为纯酶，纯酶用浓度用碧云天BCA蛋白浓度试剂盒(P0012)测定，后续纯酶的用量以蛋白含量计。
实施例3：酶活的测定
0.4mg实施例2方法制备的粗酶液或0.05mg纯酶、终浓度1.6g/L的聚磷酸(PPA)，终浓度2.25mM的磷酸腺苷(AMP)，终浓度10mM的MgCl2加入到10mL 50mM的磷酸钾缓冲液中(pH7.5)。反应液在37℃孵育5min后加入10mL 0.2M磷酸水溶液终止反应。最终溶液中的ATP含量用HPLC方法测定。
HPLC所用的仪器为Agilent 1260InfinityⅡ(安捷伦科技有限公司，美国)，配置Agilent 2414紫外检测器，Agilent 1525泵，Agilent 717进样器。色谱柱为XBridge C18column(C18,5μm,4.6×250mm,Waters,California,USA)。流动相速率为1mL/min，紫外检测波长为254nm，流动相为磷酸钾缓冲液(50mM，pH 7.0)，进样量为10μL，检测时间为9min。以不同浓度的ATP(0.125mM、0.25mM、0.5mM、1.0mM、2.0mM和4.0mM)进样得到的峰面积数据，得到ATP浓度与峰面积标准曲线，曲线方程为y＝(x+189.78)/3847(R2＝0.998)，其中y为ATP浓度(mM)，x为液相得到ATP峰面积。
酶活定义：一个酶活定义为上述条件下每分钟产生1μmol ATP所需的酶量(前5分钟)。
实施例2中0.4mg粗酶液与0.05mg纯酶液的酶活分别是0.0029U与0.0932U。
实施例4：丙氨酸突变鉴定ChPPK中关键位点
1、突变位点的筛选
用含有5个磷酸的polyP代表底物PPA。使用Autodock vina 1.1.2进行ChPPK与底物的模拟(AMP与5个磷酸的polyP)对接。根据酶与底物结合的结果，选取大部分与底物距离在(D77、G80、K81、D82、F102、K103、V104、P105、R117、R133、E137、N138、V141和R208)以及部分与底物距离在/>(S106、I108、S111)的氨基酸。采用实施例1中的pET28-ChPPK质粒为模板，采用Quick-change的突变方法，利用表1中列举的引物，将各个位点氨基酸突变成丙氨酸。突变PCR体系含有25μl 2×Phanta Max混合液，上下游引物各0.4μM，约10ng模板，最后补纯水到50μl。PCR条件为：95℃1min。然后20个扩增循环，每个扩增循环包括95℃10s，55℃30s，72℃5min。最后72℃5min。
表1、突变位点及引物
2、突变工程菌及粗酶液
将步骤1突变的质粒转化宿主菌E.coli BL21(DE3)，采用实施例2方法制备粗酶液，按实施例3方法测定相对酶活。结果如图1所示，以野生型粗酶液的活力为100％，D77、D82、R133、E137和R208残基突变成丙氨酸后，含有ChPPK突变体的裂解液上清彻底失去活力，说明这些位点为保守氨基酸，不适合进行进化研究。G80、K81、F102、K103、P105、S106、I108、S111和R117突变后酶活发生了一定程度的改变，证明这些位点对ChPPK活力影响较大，所以这些位点在实施例5中进行了饱和突变研究。
实施例5：饱和突变提高ChPPK的活力
1、定点饱和突变
除了实施例4的候选位点外，A79与L285也被选择用于饱和突变，理由如下：在ChPPK与底物结合模型中，A79位点与polyP的距离在范围之内。由于本身序列为丙氨酸，所以在实施例4中没有进行丙氨酸突变。在实施例5中，直接对A79进行了饱和突变。另外，L285是决定ChPPK盖子结构的关键位点，所以在实施例5中也进行了饱和突变。
采用Quick-change的突变方法，以实施例1方法构建的pET28-ChPPK质粒为模板，采用表2引物，选取位点A79、G80、K81、F102、K103、P105、S106、I108、S111、R117、R208和L285进行饱和突变。
表2、突变位点及引物
表2中N＝A，T，G，C；K＝G，T；M＝A，C。
2、突变工程菌及粗酶液
将步骤1突变的质粒转化宿主菌E.coli BL21(DE3)，采用实施例2方法制备纯酶，按实施例3方法测定酶活。
结果如图2所示，含有A79G、S106C、I108F、I108N、I108Y、S111E、S111K、S111A或L285P的单残基突变体酶活有显著提升。
实施例6：有益突变体组合提高ChPPK的活力
1、双突变、三突变
单残基突变酶活提高的位点位于ChPPK氨基酸序列的79、106、108、111以及285位上。根据酶与底物对接结果，79、106、108和111位于底物结合口袋，而285位点距离这些底物结合位点较远。所以285位点与其他有益突变产生相互影响的可能性较小。因此首先对79、106、108和111位进行了组合突变。为了将79、106、108和111位点的有益突变进行组合，我们采用PCR的方法得到一段含有79、106、108和111位所有有益突变可能的片段。该PCR采用pET28-ChPPK质粒为模板，PPK-M-b primer F、PPK-M-b primer R1、PPK-M-b primer R2为引物。其中PPK-M-b primer F为上游引物，PPK-M-b primer R1与PPK-M-b primer R2的混合物(PCR反应中加入摩尔比1：1)为下游引物。上下游引物中包含了兼并碱基，可以产生所有可能的有益突变。
PPK-M-b primer F：TCAGGCAATGGATGCAGSAGGTAAAGATGGTA；
PPK-M-b primer R1：ACAGATAATCATGCTYCAGTTCAWWTTTASACGGAAC。
PPK-M-b primer R2：ACAGATAATCATGTGMCAGTTCAWWTTTASACGGAAC
兼并碱基(S＝G，C；Y＝C，T；W＝A，T；M＝A，C)
为了将这个含有79、106、108和111位所有可能有益突变的片段重新连接到质粒中，用PCR扩增了不包含这个片段的pET28-ChPPK质粒。引物如下：
pET-PPK primer F：CATGATTATCTGTGGCGTCATTATGTG；
pET-PPK primer R：TGCATCCATTGCCTGAAAAACAATCAG。
将这个质粒片段与含有79、106、108和111位所有有益突变可能的片段链接后转化宿主菌E.coli BL21(DE3)，采用实施例2方法制备纯酶，按实施例3方法测定酶活。结果如图3所示，带有A79G/S108F的二突变ChPPK和带有A79G/S106C/I108F的三突变ChPPK有着较高的酶活。带有I108F/S111E的二突变和带有S106C/I108Y/S111K的三突变的活力较低。
2、四突变
将带有A79G/S106C/I108F的三位点突变的pET-ChPPK进行进一步突变，引入有益突变S111A突变。采用Quick-change的突变方法，以含有A79G/S106C/I108F的三位点突变的pET28-ChPPK质粒为模板，采用表1中111位点丙氨酸突变扩增引物进突变。将上述突变的质粒转化宿主菌E.coli BL21(DE3)后，如实施例1、2和3进行筛选、表达和纯化。结果如图3所示，带有A79G/S106C/I108F/S111A的四位点突变ChPPK活力较带有A79G/S106C/I108F的三位点突变的ChPPK明显下降。为了检测S111位点其他有益突变叠加对A79G/S106C/I108F的三位点突变的pET28-ChPPK的影响，采用Quick-change的突变方法，以含有A79G/S106C/I108F的三位点突变的pET28-ChPPK质粒为模板，采用如下引物将111位点突变成谷氨酸。
将111位的丝氨酸突变为谷氨酸所用的引物：
正向引物：TTGAACTGGAACATGATTATCTGTGGC；
反向引物：TAATCATGTTCCAGTTCAATTTTGGACG。
产生带有A79G/S106C/I108F/S111K的四突变ChPPK较带有A79G/S106C/I108F/的三突变ChPPK活力明显下降。说明把79、106、108和111位所有有益突变叠加并不能产生最佳效果。
将带有A79G/S106C/I108F的三位点突变的pET-ChPPK进行进一步突变，引入L285P突变。采用Quick-change的突变方法，以含有A79G/S106C/I108F的三位点突变的pET28-ChPPK质粒为模板，采用下列引物进突变。将上述突变的质粒转化宿主菌BL21(DE3)后，如实施例1、2和3进行筛选、表达和纯化。结果图3所示，带有A79G/S106C/I108F/L285P的四位点联合突变体(ChPPK/A79G/S106C/I108F/L285P)具有最高的活性。
285位点突变
正向引物：ACCGTGAGCCCAGAACAGAAAGCGG；
反向引物：TTCTGTTCTGGGCTCACGGTCGGAAA。
实施例7：突变体ChPPK/A79G/S106C/I108F/L285P的特性分析
我们比较了温度、pH和底物浓度对野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P活性的影响。
1、温度
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P，采用实施例2方法制备纯酶。采用实施例3方法测酶活，测酶活的温度分别改为25℃、30℃、35℃、37℃、40℃、45℃、50℃、55℃或60℃。
结果见图4所示，虽然37℃是野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P的最佳反应温度，但突变型ChPPK/A79G/S106C/I108F/L285P在42-50℃温度下表现出显著更高的相对活性。例如，突变体ChPPK/A79G/S106C/I108F/L285P在45℃时保留了82％的活性，而野生型ChPPK在这种条件下失去了96％的活性。
2、pH
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P采用实施例2方法制备纯酶。采用实施例3方法测酶活，并将缓冲液pH分别改为5.0-6.0(50mM柠檬酸-柠檬酸钠缓冲液)，6.0-8.0(50mM磷酸钾缓冲液)，8.0-9.0(50mM硼砂-硼酸缓冲液)或9.0-10.0(50mM甘氨酸-NaOH缓冲液)。
结果见图5所示，野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P的相对活性在pH值为7.5时达到最大值。然而，突变体ChPPK/A79G/S106C/I108F/L285P在酸性条件下具有较高的相对活性。
3、AMP浓度
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P采用实施例2方法制备纯酶。采用实施例3方法测酶活，并将AMP浓度分别改为0.25mM、0.50mM、0.75mM、1.00mM、1.50mM、2.00mM、2.50mM、3.00mM、3.50mM、4.00mM、4.50mM或5.00mM。
结果见图6所示，野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P在AMP浓度为2.0-2.5mM时达到了最高的相对活性。
4、PPA浓度
将实施例1和6方法构建的工程菌E.coli BL21(DE3)-ChPPK和E.coli BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P采用实施例2方法制备纯酶。采用实施例3方法测酶活，并将PPA浓度分别改为0.32g/L、0.64g/L、0.96g/L、1.28g/L、1.60g/L、1.92g/L、2.24g/L、2.56g/L、2.88g/L或3.20g/L。
结果见图7所示，野生型ChPPK的最佳PPA浓度为1.6g/L，而突变型ChPPK/A79G/S106C/I108F/L285P在2.24g/L PPA存在下表现出最高的相对活性，表明突变型ChPPK/A79G/S106C/I108F/L285P具有更高的PPA耐受性。
实施例8：用纯酶测定野生型ChPPK和突变型A79G/S106C/I108F/L285P的动力学参数
将实施例1构建的野生型Ecoli.BL21(DE3)-ChPPK和实施例6方法构建的工程菌Ecoli.BL21(DE3)-ChPPK/A79G/S106C/I108F/L285P，采用实施例2方法分别制备野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P的纯酶。采用pseudo-one-substrate动力学模型计算Km值与Kcat值。为了分别计算酶对双底物的动力学，采用实施例3方法测试酶活，但反应在固定PPA浓度(1.6g/L)的条件下调整AMP浓度(0、0.25、0.5、0.75、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0mM)或在固定AMP浓度(5mM)的条件下调整PPA浓度(0.32、0.64、0.96、1.28、1.6、1.92、2.24、2.56、2.88、3.2g/L)条件下进行。
野生型ChPPK对AMP和PPA的Km值与突变型ChPPK/A79G/S106C/I108F/L285P的Km值相似(表3和表4)。突变型ChPPK/A79G/S106C/I108F/L285P的酶活性的提高可以解释为其对AMP和PPA的周转数(kcat值)的增加。突变型ChPPK/A79G/S106C/I108F/L285P对AMP和PPA的催化效率(kcat/Km)分别是野生型ChPPK的16倍和18倍。
表3、野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P对底物AMP的动力学参数
表4、野生型ChPPK和突变型ChPPK/A79G/S106C/I108F/L285P对底物PPA的动力学参数
实施例9：突变体ChPPK/A79G/S106C/I108F/L285P在NMN生物合成中的应用
1、烟酰胺核糖激酶粗酶液
由于烟酰胺核糖激酶可以催化烟酰胺核糖(NR)生物合成烟酰胺单核苷酸(NMN)，因此以来自棕硬尾鸭(Oxyura jamaicensis)的烟酰胺核糖激酶(NRK，GenBank号：XP_035204248.1)氨基酸序列为模板，人工合成针对大肠杆菌密码子优化了的烟酰胺核糖激酶基因，核苷酸序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示。
SEQ ID NO:3
ATGAAATACATCATCGGTATCGGTGGTGTTACCAACGGTGGCAAAACCACCCTGACAAATCGTCTGGTTAAAGCACTGCCTAACTGTTGTGTGGTTCACCAGGACGATTTTTTTAAACCTCAGGATCAGATTGAAGTTGGTGAAGATGGCTTTAAACAATGGGACGTTCTGGACTCTCTGGATATGGAAGCAATGGTTAGCACCGTTCGTGCATGGATTGAAAATCCGGTTAAATTTGCACGTAGCCACGGTGTTAATGTTACACCGGGCAGCAAAGAACCGGCAAGCAAAGATATTCATATTCTGGTTATTGAGGGATTTCTGCTGTATAATTATAAACCGCTGATTGACCTGTTTGATATTCGTTATTATCTGGCAGTCCCTTATGATGAATGTAAACGTCGTCGTAGCACCCGTAACTATACCGTTCCGGATCCGCCGGGTCTGTTCGATGGCCATGTTTGGCCGATGTATCTGAAACATCGTAAAGAAATGGAAGACAATGGGGTGGATGTGGTTTATCTGGATGGCCTGAAAAGCCGCGATGAACTGTACAACCAGGTCTTTGAAGATATTCAGAATAAACTGCTGAACTGCCTGCATCATCATCACCACCATTAA。
SEQ ID NO:4
MKYIIGIGGVTNGGKTTLTNRLVKALPNCCVVHQDDFFKPQDQIEVGEDGFKQWDVLDSLDMEAMVSTVRAWIENPVKFARSHGVNVTPGSKEPASKDIHILVIEGFLLYNYKPLIDLFDIRYYLAVPYDECKRRRSTRNYTVPDPPGLFDGHVWPMYLKHRKEMEDNGVDVVYLDGLKSRDELYNQVFEDIQNKLLNCLHHHHHH。
将针对大肠杆菌密码子优化了的重组基因NRK(SEQ ID NO:3所示)插入到pET-28a(+)的T7启动子下，得到表达质粒pET28-NRK。将该表达质粒转化至大肠杆菌Ecoli.BL21(DE3)中，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下培养8-12h，挑取阳性克隆，即为野生型E.coli BL21(DE3)-NRK，用于表达重组NRK。与实施例2中相同条件制备粗酶液，其中的NRK的使用量以对应破碎前菌体量计。
2、生物合成NMN
反应1(25mM ATP)：在1mL磷酸钾缓冲液(50mM，pH 6.5)中加入终浓度25mM的ATP、终浓度100mM的NR、终浓度10mM氯化镁和8mg/mL的NRK粗酶液，在37℃反应6h，在0.5h、1.5h、3h和6h取样，采用实例3中HPLC方法检测(NMN保留时间为2.9分钟)。NMN浓度与峰面积标准曲线获得方法：以不同浓度的NMN(0.0625mM、0.125mM、0.25mM、0.5mM、1.0mM和2.0mM)进样得到的峰面积数据，得到NMN浓度与峰面积标准曲线，曲线方程为y＝(x+254.3)/4587(R2＝0.995)，其中y为NMN的浓度(mM)，x为NMN的峰面积。反应1生成的NMN浓度变化如图8所示，6小时后NMN的产量为34mM。
反应2(100mM ATP)：将反应1中ATP浓度改为100mM，其他操作相同，结果见图8所示，当投入的ATP达到100mM时，6小时后NMN的产量提升到85mM。
反应3(25mM ATP+野生型ChPPK)：在反应1中加入4mg/mL的实施例2方法制备的野生型ChPPK粗酶液和4.8g/L的PPA，其他操作相同，NMN浓度变化如图8所示，当含有野生型ChPPK ATP再生系统被引入NMN合成，6小时后NMN的产量提升到55mM。
反应4(25mM ATP+突变体ChPPK/A79G/S106C/I108F/L285P)：在反应1中加入4mg/mL的实施例6方法制备的突变体ChPPK/A79G/S106C/I108F/L285P粗酶液和4.8g/L的PPA，其他操作相同，结果见图8所示，当含有突变体ChPPK/A79G/S106C/I108F/L285P的ATP再生系统被引入NMN合成，6小时后NMN的产量提升到86mM。说明导入突变体构成的ATP再生系统，可以节省75％的ATP投入量而不影响最终NMN的产量。
实施例10：突变体ChPPK/A79G/S106C/I108F/L285P在G6P生物合成中的应用1、己糖激酶粗酶液
由于己糖激酶可以催化葡萄糖生物合成葡萄糖6磷酸(G6P)，因此以来自酿酒酵母(Saccharomyces cerevisiae)的己糖激酶(HK，GenBank号：NP_013551.1)氨基酸序列为模板，人工合成针对大肠杆菌密码子优化了的己糖激酶基因，核苷酸序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示。
SEQ ID NO:5
ATGACCATTGAAAGCACCCTGGCACGCGAACTGGAAAGTCTGATTCTGCCGGCGGATAGCATTGTGAATGTGGTGGATCAGTTTCAGGAAGAACTGCTGAGCCGCCTGCAGACCAACACCATTAGCATGCTGCCGCAGTGCCTGGTGCCGGATAAACGCAGCCGCTGGAATCCGGAAGATAAAATTCTGACCATTGATTTTGGTGGTACCCGTCTGAAATTTGCGATTATTAGCCTTCCGCAGATTGTGATTGAATACAACGATGCGTTTGAACTGACCTATAACATTGTGGATTCAAATTTCTTTAACCAGATCATTTATACCATTTGCACCCGCCTGGCCGCCAATGGTTATATCAAAAAAAAAAACGAAAGCTCAGAAGCGTCAAAATTTTTTGTGAGCGTGACCTTTAGCTTTCCGCTGAACCCGGAAGGCGAAGTGGTGGCGATGGGCAAAGGTTTTGTGATGACCGATACCCTGCAGGGCAGCACCGTGAAACAGCTGATTCAGAGCAGCTTTCATCGCATTATTAGCGAGAATATTGAAGAGTTTTTTTGCACCATGAATGTGTGTCATGTGATTAATGATGCCATTGCCGTGAGCCTGACCAGCAAATTTATTTGTGAAAACGATAGCATCAGCCTGATTATTGGCACCGGTACCAATGCGTGCTTTGAAGTGCCGTATGGCTATCTGCCGCCGTTTAAACGCGATGCGCTGCGCGAAACCCTGCCGAGCAGCTACAACAAAGAAACCCTGAATTTTAAACATGTGCTGATCAACAGCGAAATCGGCTTTATTGGCAAAAATGTCATTGCGCTGCAGCCGTTTGATATTCACGGCGCAATTAGCTATGAAATGCCGCTGGAATGCGTGACCAGCGGCAAATGGCTGCCGCTGAGCCTGAAAAACATTCTGCTGCAATATAATATTATTCCGAAAAATTTTCCGGTTGAATTTAATGGAGAACTGGTGTGCCAGCTGGCGGAAGATTGCACCAATGCGTGGTTTGAAAATGAACATTATGCCCTGATTTGCCAGATTGCGCGCCTGTTGATTAAACGCGCAGCGTTCTACGTGGCGGCCATTGTGCAGGCGATTGATATTATCACCGGCTGCAAAAATTATAATTTTATTCACATTGGCTATGTGGGCTCATTTCTGCATAACAGCAACTTTTACCGTGAACAGATTAAATATTATAGCAGCATTCACATTAAACTGCAGTTCCTGAATCACTCAAATCTGCTGGGTGCGGCCATTGCCACCTACCTGAATAAATCAGATAACCAGGTGCAGTAA
SEQ ID NO:6
MTIESTLARELESLILPADSIVNVVDQFQEELLSRLQTNTISMLPQCLVPDKRSRWNPEDKILTIDFGGTRLKFAIISLPQIVIEYNDAFELTYNIVDSNFFNQIIYTICTRLAANGYIKKKNESSEASKFFVSVTFSFPLNPEGEVVAMGKGFVMTDTLQGSTVKQLIQSSFHRIISENIEEFFCTMNVCHVINDAIAVSLTSKFICENDSISLIIGTGTNACFEVPYGYLPPFKRDALRETLPSSYNKETLNFKHVLINSEIGFIGKNVIALQPFDIHGAISYEMPLECVTSGKWLPLSLKNILLQYNIIPKNFPVEFNGELVCQLAEDCTNAWFENEHYALICQIARLLIKRAAFYVAAIVQAIDIITGCKNYNFIHIGYVGSFLHNSNFYREQIKYYSSIHIKLQFLNHSNLLGAAIATYLNKSDNQVQ.
将针对大肠杆菌密码子优化了的重组基因HK(核苷酸序列如SEQ ID NO:5所示)插入到pET-28a(+)的T7启动子下，得到表达质粒pET28-HK。将该表达质粒转化至大肠杆菌Ecoli.BL21(DE3)中，涂布于含有50μg/mL卡那霉素抗性的LB平板，37℃下培养8-12h，挑取阳性克隆，即为野生型E.coli BL21(DE3)-HK，用于表达重组HK。与实施例2中相同条件制备粗酶液，其中HK粗酶液的使用量以对应破碎前菌体量计。
2、生物合成G6P
反应1(25mM ATP)：在5mL磷酸钾缓冲液(50mM，pH 7.5)中加入终浓度25mM ATP、终浓度100mM葡萄糖、终浓度10mM氯化镁和12mg/mL的HK粗酶液，在30℃反应8h。反应结束后取样1mL，70℃加热15分钟使酶失活。12,000×g离心10分钟取上清进行HPLC分析。
HPLC检测G6P的仪器为Agilent 1260InfinityⅡ(安捷伦科技有限公司，美国)，配置Dionex ED40 detector检测器。Agilent 1525泵，Agilent 717进样器，色谱柱为DionexIonPac AS11-HC，检测温度为30℃。流动相为氢氧化钠水溶液，速率为1mL/min。采用梯度洗脱的方法：0-10分钟内为25mM氢氧化钠水溶液，12-15分钟内氢氧化钠水溶液从25mM提升到100mM，最后17-21分钟换成25mM氢氧化钠水溶液。G6P浓度与峰面积标准曲线获得方法：以不同浓度的G6P水溶液(0.125mM、0.25mM、0.5mM、1.0mM、2.0mM和4.0mM)进样得到的峰面积数据，得到G6P浓度与峰面积标准曲线，曲线方程为y＝(x+544.7)/3856(R2＝0.997)，其中y为G6P的浓度(mM)，x为G6P的峰面积。用该标准曲线计算HK粗酶液催化生成的G6P产量。如图9所示，8h后G6P的产量为18mM。
反应2(100mM ATP)：将反应1中ATP浓度改为100mM，其他操作相同，结果见图9所示，当投入的ATP达到100mM时，8小时后G6P的产量提升到78mM。
反应3(25mM ATP+野生型ChPPK)：在反应1中加入4mg/mL实施例2方法制备的野生型ChPPK粗酶液和4.8g/L的PPA，其他操作相同。如图9所示，当含有野生型ChPPK ATP再生系统被引入G6P合成，8小时后G6P的产量提升到38mM。
反应4(25mM ATP+突变体ChPPK/A79G/S106C/I108F/L285P)：在反应1中加入4mg/mL实施例6方法制备的突变体ChPPK/A79G/S106C/I108F/L285P粗酶液和4.8g/L的PPA，其他操作相同，结果见图9所示，当含有突变体ChPPK/A79G/S106C/I108F/L285P的ATP再生系统被引入G6P合成，8小时后G6P的产量提升到82mM。说明导入突变体构成的ATP再生系统，可以节省75％的ATP投入量而不影响最终G6P的产量。
序列表
<110> 浙江工业大学
<120> 一种多聚磷酸激酶突变体、工程菌及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 933
<212> DNA
<213> 哈氏嗜纤维菌(Cytophaga hutchinsonii)
<400> 1
atggcaaccg attttagcaa actgagcaaa tatgttgaaa cgctgcgtgt gaaaccgaaa 60
cagagcattg atctgaaaaa ggattttgat accgattatg atcataaaat gctgacgaaa 120
gaagaaggtg aagaactgct gaatctgggt attagtaaac tgagcgaaat tcaggaaaaa 180
ctgtatgcat ctggcacaaa aagcgtgctg attgtttttc aggcaatgga tgcagcaggt 240
aaagatggta ccgttaaaca tattatgacg ggtctgaatc cgcagggtgt taaagtgacc 300
agctttaaag ttccgtccaa aattgaactg agtcatgatt atctgtggcg tcattatgtg 360
gcactgccgg caaccggcga aattggtatt tttaaccgta gccattatga aaatgtgctg 420
gttacccgtg tacatccgga atatctgctg agcgaacaga ccagcggtgt taccgcaatt 480
gaacaggtaa atcagaaatt ttgggataaa cgctttcagc agatcaataa ctttgaacag 540
catattagcg aaaacggtac cattgttctg aaattttttc tgcatgtttc caaaaaggaa 600
cagaaaaagc gttttattga acgtatcgaa ctggatacca aaaattggaa attttcaacc 660
ggtgatctga aagaacgtgc ccattggaaa gattatcgta atgcgtatga agatatgctg 720
gcaaatacct ctaccaaaca ggccccgtgg tttgttattc cggccgatga taaatggttt 780
acccgtctgc tgattgcaga aattatctgt accgaactgg aaaaactgaa tctgaccttt 840
ccgaccgtga gcctggaaca gaaagcggaa ctggaaaaag caaaagcaga actggttgca 900
gaaaaatcaa gcgatcatca tcatcaccac taa 933
<210> 2
<211> 311
<212> PRT
<213> 哈氏嗜纤维菌(Cytophaga hutchinsonii)
<400> 2
Met Ala Thr Asp Phe Ser Lys Leu Ser Lys Tyr Val Glu Thr Leu Arg
1 5 10 15
Val Lys Pro Lys Gln Ser Ile Asp Leu Lys Lys Asp Phe Asp Thr Asp
20 25 30
Tyr Asp His Lys Met Leu Thr Lys Glu Glu Gly Glu Glu Leu Leu Asn
35 40 45
Leu Gly Ile Ser Lys Leu Ser Glu Ile Gln Glu Lys Leu Tyr Ala Ser
50 55 60
Gly Thr Lys Ser Val Leu Ile Val Phe Gln Ala Met Asp Ala Ala Gly
65 70 75 80
Lys Asp Gly Thr Val Lys His Ile Met Thr Gly Leu Asn Pro Gln Gly
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Val Lys Val Thr Ser Phe Lys Val Pro Ser Lys Ile Glu Leu Ser His
100 105 110
Asp Tyr Leu Trp Arg His Tyr Val Ala Leu Pro Ala Thr Gly Glu Ile
115 120 125
Gly Ile Phe Asn Arg Ser His Tyr Glu Asn Val Leu Val Thr Arg Val
130 135 140
His Pro Glu Tyr Leu Leu Ser Glu Gln Thr Ser Gly Val Thr Ala Ile
145 150 155 160
Glu Gln Val Asn Gln Lys Phe Trp Asp Lys Arg Phe Gln Gln Ile Asn
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Asn Phe Glu Gln His Ile Ser Glu Asn Gly Thr Ile Val Leu Lys Phe
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Phe Leu His Val Ser Lys Lys Glu Gln Lys Lys Arg Phe Ile Glu Arg
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Ile Glu Leu Asp Thr Lys Asn Trp Lys Phe Ser Thr Gly Asp Leu Lys
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Glu Arg Ala His Trp Lys Asp Tyr Arg Asn Ala Tyr Glu Asp Met Leu
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Ala Asn Thr Ser Thr Lys Gln Ala Pro Trp Phe Val Ile Pro Ala Asp
245 250 255
Asp Lys Trp Phe Thr Arg Leu Leu Ile Ala Glu Ile Ile Cys Thr Glu
260 265 270
Leu Glu Lys Leu Asn Leu Thr Phe Pro Thr Val Ser Leu Glu Gln Lys
275 280 285
Ala Glu Leu Glu Lys Ala Lys Ala Glu Leu Val Ala Glu Lys Ser Ser
290 295 300
Asp His His His His His His
305 310
<210> 3
<211> 621
<212> DNA
<213> 棕硬尾鸭(Oxyura jamaicensis)
<400> 3
atgaaataca tcatcggtat cggtggtgtt accaacggtg gcaaaaccac cctgacaaat 60
cgtctggtta aagcactgcc taactgttgt gtggttcacc aggacgattt ttttaaacct 120
caggatcaga ttgaagttgg tgaagatggc tttaaacaat gggacgttct ggactctctg 180
gatatggaag caatggttag caccgttcgt gcatggattg aaaatccggt taaatttgca 240
cgtagccacg gtgttaatgt tacaccgggc agcaaagaac cggcaagcaa agatattcat 300
attctggtta ttgagggatt tctgctgtat aattataaac cgctgattga cctgtttgat 360
attcgttatt atctggcagt cccttatgat gaatgtaaac gtcgtcgtag cacccgtaac 420
tataccgttc cggatccgcc gggtctgttc gatggccatg tttggccgat gtatctgaaa 480
catcgtaaag aaatggaaga caatggggtg gatgtggttt atctggatgg cctgaaaagc 540
cgcgatgaac tgtacaacca ggtctttgaa gatattcaga ataaactgct gaactgcctg 600
catcatcatc accaccatta a 621
<210> 4
<211> 206
<212> PRT
<213> 棕硬尾鸭(Oxyura jamaicensis)
<400> 4
Met Lys Tyr Ile Ile Gly Ile Gly Gly Val Thr Asn Gly Gly Lys Thr
1 5 10 15
Thr Leu Thr Asn Arg Leu Val Lys Ala Leu Pro Asn Cys Cys Val Val
20 25 30
His Gln Asp Asp Phe Phe Lys Pro Gln Asp Gln Ile Glu Val Gly Glu
35 40 45
Asp Gly Phe Lys Gln Trp Asp Val Leu Asp Ser Leu Asp Met Glu Ala
50 55 60
Met Val Ser Thr Val Arg Ala Trp Ile Glu Asn Pro Val Lys Phe Ala
65 70 75 80
Arg Ser His Gly Val Asn Val Thr Pro Gly Ser Lys Glu Pro Ala Ser
85 90 95
Lys Asp Ile His Ile Leu Val Ile Glu Gly Phe Leu Leu Tyr Asn Tyr
100 105 110
Lys Pro Leu Ile Asp Leu Phe Asp Ile Arg Tyr Tyr Leu Ala Val Pro
115 120 125
Tyr Asp Glu Cys Lys Arg Arg Arg Ser Thr Arg Asn Tyr Thr Val Pro
130 135 140
Asp Pro Pro Gly Leu Phe Asp Gly His Val Trp Pro Met Tyr Leu Lys
145 150 155 160
His Arg Lys Glu Met Glu Asp Asn Gly Val Asp Val Val Tyr Leu Asp
165 170 175
Gly Leu Lys Ser Arg Asp Glu Leu Tyr Asn Gln Val Phe Glu Asp Ile
180 185 190
Gln Asn Lys Leu Leu Asn Cys Leu His His His His His His
195 200 205
<210> 5
<211> 1302
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 5
atgaccattg aaagcaccct ggcacgcgaa ctggaaagtc tgattctgcc ggcggatagc 60
attgtgaatg tggtggatca gtttcaggaa gaactgctga gccgcctgca gaccaacacc 120
attagcatgc tgccgcagtg cctggtgccg gataaacgca gccgctggaa tccggaagat 180
aaaattctga ccattgattt tggtggtacc cgtctgaaat ttgcgattat tagccttccg 240
cagattgtga ttgaatacaa cgatgcgttt gaactgacct ataacattgt ggattcaaat 300
ttctttaacc agatcattta taccatttgc acccgcctgg ccgccaatgg ttatatcaaa 360
aaaaaaaacg aaagctcaga agcgtcaaaa ttttttgtga gcgtgacctt tagctttccg 420
ctgaacccgg aaggcgaagt ggtggcgatg ggcaaaggtt ttgtgatgac cgataccctg 480
cagggcagca ccgtgaaaca gctgattcag agcagctttc atcgcattat tagcgagaat 540
attgaagagt ttttttgcac catgaatgtg tgtcatgtga ttaatgatgc cattgccgtg 600
agcctgacca gcaaatttat ttgtgaaaac gatagcatca gcctgattat tggcaccggt 660
accaatgcgt gctttgaagt gccgtatggc tatctgccgc cgtttaaacg cgatgcgctg 720
cgcgaaaccc tgccgagcag ctacaacaaa gaaaccctga attttaaaca tgtgctgatc 780
aacagcgaaa tcggctttat tggcaaaaat gtcattgcgc tgcagccgtt tgatattcac 840
ggcgcaatta gctatgaaat gccgctggaa tgcgtgacca gcggcaaatg gctgccgctg 900
agcctgaaaa acattctgct gcaatataat attattccga aaaattttcc ggttgaattt 960
aatggagaac tggtgtgcca gctggcggaa gattgcacca atgcgtggtt tgaaaatgaa 1020
cattatgccc tgatttgcca gattgcgcgc ctgttgatta aacgcgcagc gttctacgtg 1080
gcggccattg tgcaggcgat tgatattatc accggctgca aaaattataa ttttattcac 1140
attggctatg tgggctcatt tctgcataac agcaactttt accgtgaaca gattaaatat 1200
tatagcagca ttcacattaa actgcagttc ctgaatcact caaatctgct gggtgcggcc 1260
attgccacct acctgaataa atcagataac caggtgcagt aa 1302
<210> 6
<211> 433
<212> PRT
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 6
Met Thr Ile Glu Ser Thr Leu Ala Arg Glu Leu Glu Ser Leu Ile Leu
1 5 10 15
Pro Ala Asp Ser Ile Val Asn Val Val Asp Gln Phe Gln Glu Glu Leu
20 25 30
Leu Ser Arg Leu Gln Thr Asn Thr Ile Ser Met Leu Pro Gln Cys Leu
35 40 45
Val Pro Asp Lys Arg Ser Arg Trp Asn Pro Glu Asp Lys Ile Leu Thr
50 55 60
Ile Asp Phe Gly Gly Thr Arg Leu Lys Phe Ala Ile Ile Ser Leu Pro
65 70 75 80
Gln Ile Val Ile Glu Tyr Asn Asp Ala Phe Glu Leu Thr Tyr Asn Ile
85 90 95
Val Asp Ser Asn Phe Phe Asn Gln Ile Ile Tyr Thr Ile Cys Thr Arg
100 105 110
Leu Ala Ala Asn Gly Tyr Ile Lys Lys Lys Asn Glu Ser Ser Glu Ala
115 120 125
Ser Lys Phe Phe Val Ser Val Thr Phe Ser Phe Pro Leu Asn Pro Glu
130 135 140
Gly Glu Val Val Ala Met Gly Lys Gly Phe Val Met Thr Asp Thr Leu
145 150 155 160
Gln Gly Ser Thr Val Lys Gln Leu Ile Gln Ser Ser Phe His Arg Ile
165 170 175
Ile Ser Glu Asn Ile Glu Glu Phe Phe Cys Thr Met Asn Val Cys His
180 185 190
Val Ile Asn Asp Ala Ile Ala Val Ser Leu Thr Ser Lys Phe Ile Cys
195 200 205
Glu Asn Asp Ser Ile Ser Leu Ile Ile Gly Thr Gly Thr Asn Ala Cys
210 215 220
Phe Glu Val Pro Tyr Gly Tyr Leu Pro Pro Phe Lys Arg Asp Ala Leu
225 230 235 240
Arg Glu Thr Leu Pro Ser Ser Tyr Asn Lys Glu Thr Leu Asn Phe Lys
245 250 255
His Val Leu Ile Asn Ser Glu Ile Gly Phe Ile Gly Lys Asn Val Ile
260 265 270
Ala Leu Gln Pro Phe Asp Ile His Gly Ala Ile Ser Tyr Glu Met Pro
275 280 285
Leu Glu Cys Val Thr Ser Gly Lys Trp Leu Pro Leu Ser Leu Lys Asn
290 295 300
Ile Leu Leu Gln Tyr Asn Ile Ile Pro Lys Asn Phe Pro Val Glu Phe
305 310 315 320
Asn Gly Glu Leu Val Cys Gln Leu Ala Glu Asp Cys Thr Asn Ala Trp
325 330 335
Phe Glu Asn Glu His Tyr Ala Leu Ile Cys Gln Ile Ala Arg Leu Leu
340 345 350
Ile Lys Arg Ala Ala Phe Tyr Val Ala Ala Ile Val Gln Ala Ile Asp
355 360 365
Ile Ile Thr Gly Cys Lys Asn Tyr Asn Phe Ile His Ile Gly Tyr Val
370 375 380
Gly Ser Phe Leu His Asn Ser Asn Phe Tyr Arg Glu Gln Ile Lys Tyr
385 390 395 400
Tyr Ser Ser Ile His Ile Lys Leu Gln Phe Leu Asn His Ser Asn Leu
405 410 415
Leu Gly Ala Ala Ile Ala Thr Tyr Leu Asn Lys Ser Asp Asn Gln Val
420 425 430
Gln
Claims (9)
1.一种多聚磷酸激酶突变体，其特征在于，所述多聚磷酸激酶突变体是将SEQ ID NO：2所示氨基酸序列突变为下列之一：（1）第79位丙氨酸突变变为甘氨酸；（2）第106位丝氨酸突变为半胱氨酸；（3）第108位异亮氨酸突变为苯丙氨酸、天冬酰胺或酪氨酸；（4）第111位丝氨酸突变为谷氨酸或赖氨酸或丙氨酸；（5）第285位亮氨酸突变为脯氨酸；（6）第79位丙氨酸突变为甘氨酸、第108位异亮氨酸突变为苯丙氨酸；（7）第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸；（8）第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第111位丝氨酸突变为丙氨酸；（9）第79位丙氨酸突变为甘氨酸、第106位丝氨酸突变为半胱氨酸、第108位异亮氨酸突变为苯丙氨酸、第285位亮氨酸突变为脯氨酸。
2.一种权利要求1所述多聚磷酸激酶突变体的编码基因。
3.一种包含权利要求2所述编码基因的重组基因工程菌。
4.一种权利要求1所述多聚磷酸激酶突变体在构建ATP再生体系中的应用。
5.一种权利要求1所述多聚磷酸激酶突变体在合成烟酰胺单核苷酸中的应用，其特征在于，所述应用的方法为：将所述多聚磷酸激酶突变体基因工程菌和烟酰胺核糖激酶基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎，以破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸和烟酰胺核糖为底物，加入氯化镁和聚磷酸，以pH6.5的缓冲液为反应介质构成反应体系，在37℃进行反应，反应液分离纯化，获得烟酰胺单核苷酸。
6.如权利要求5所述的应用，其特征在于，所述腺嘌呤核苷三磷酸加入量以缓冲液体积计为10-100 mM；所述烟酰胺核糖加入量以缓冲液体积计为50-200 mM；所述氯化镁加入量以缓冲液体积计为5-20 mM；所述聚磷酸加入量以缓冲液体积计为1-10 g/L；所述多聚磷酸激酶突变体加入量以破碎前湿菌体量计为2-30 mg/mL缓冲液；所述烟酰胺核糖激酶加入量以破碎前湿菌体量计为5-30 mg/mL缓冲液。
7.如权利要求5所述的应用，其特征在于，所述催化剂按如下方法制备：将多聚磷酸激酶突变体基因工程菌接种至含有50 μg/mL卡那霉素抗性的LB液体培养基，37 ℃，200 rpm下培养12h，再以体积浓度1％接种量接种至新鲜的含有50 μg/mL卡那霉素抗性的LB液体培养基中，于37℃，150 rpm下培养至菌体OD600达0.6，加入终浓度为0.1 mM的IPTG，28 ℃下诱导培养12 h后，4 ℃、8000 rpm离心10 min，弃去上清液，收集湿菌体沉淀；收集的湿菌体用pH为7.5的50 mM磷酸钾缓冲液重悬，并使用超声波细胞粉碎机破碎，破碎功率为50 W，每工作1 s，间歇2 s，总共破碎20 min；收集细胞裂解液，在12000 g下离心1 min，取上清液，即为粗酶液；所述烟酰胺核糖激酶基因工程菌的上清液制备方法同多聚磷酸激酶突变体基因工程菌。
8.一种权利要求1所述多聚磷酸激酶突变体在合成葡萄糖6磷酸中的应用，其特征在于所述应用的方法为：将所述多聚磷酸激酶突变体基因工程菌和己糖激酶基因工程菌分别经诱导培养获得的湿菌体用缓冲液重悬并采用超声破碎，取破碎后的上清液为催化剂，以腺嘌呤核苷三磷酸和葡萄糖为底物，加入氯化镁和聚磷酸，以pH 7.2的缓冲液为反应介质构成反应体系，在37℃进行反应，反应液分离纯化，获得葡萄糖6磷酸。
9.如权利要求8所述的应用，其特征在于，所述腺嘌呤核苷三磷酸加入量以缓冲液体积计为10-100 mM；所述葡萄糖加入量以缓冲液体积计为20-150 mM；所述氯化镁加入量以缓冲液体积计为5-20 mM；所述聚磷酸加入量以缓冲液体积计为1-10 g/L；所述多聚磷酸激酶突变体入量以破碎前湿菌体量计为2-30 mg/mL缓冲液；所述己糖激酶加入量以破碎前湿菌体量计为5-30 mg/mL缓冲液。
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