CN112280762A - 一种烟酰胺核糖激酶突变体及其编码基因和应用 - Google Patents
一种烟酰胺核糖激酶突变体及其编码基因和应用 Download PDFInfo
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- CN112280762A CN112280762A CN202011270235.4A CN202011270235A CN112280762A CN 112280762 A CN112280762 A CN 112280762A CN 202011270235 A CN202011270235 A CN 202011270235A CN 112280762 A CN112280762 A CN 112280762A
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Abstract
本发明属于生物酶工程技术领域，具体涉及一种烟酰胺核糖激酶突变体及其编码基因和应用。所述的烟酰胺核糖激酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比，在SEQ ID NO:2所示的氨基酸序列的第9位、第102位、第52位、第146位进行单突变、两两联合突变、三个联合突变或四个联合突变中的任意一种突变。本发明所提供的烟酰胺核糖激酶突变体可用于β‑烟酰胺单核苷酸的合成制备。本发明构建的烟酰胺核糖激酶突变体与野生型酶相比，酶活力提高了3.86‑4.53倍，缩短反应时间，能显著降低酶的使用量，降低发酵容量和成本，提高NMN收率，具有大规模工业应用的广泛前景。
Description
技术领域
本发明涉及生物酶工程技术领域，具体涉及一种烟酰胺核糖激酶突变体及其编码基因和应用。
背景技术
烟酰胺单核苷酸(Nicotinamide mononuclotide，NMN)，是一种自然存在的生物活性核苷酸，NMN有2种不规则存在形式，α异构体和β异构体。其中，β异构体是NMN的活性形式，分子量为334.221g/mol。NMN是哺乳动物体内烟酰胺腺嘌呤二核苷酸(Nicotinamideadenine dinucleotide，NAD+，又称辅酶I)合成途径的重要中间体。近年来在Science、Nature、Cell等国际权威学术杂志上的相关研究报道表明，补充NMN可有效地增加和恢复体内辅酶I水平，大幅延缓衰老和防止老年痴呆症等多种神经元退化疾病，并由此从根本上调理和改善衰老的各种症状。因此以NMN为活性成分的功能性保健食品有着很大的开发潜力和市场前景。目前NMN在欧、美、日等发达国家已批准作为保健食品原料，并以NMN为主要成份开发出多种保健品，如美国HERBALmax、基因港GeneHarbor NMN9000、日本MIRAI LABNMN3000胶囊等。
NMN目前的生产方法主要有三种：固态酵母发酵工艺、体外酶催化工艺和化学法合成工艺。其中：(1)固态酵母发酵工艺复杂，产量较低，因此产品价格高昂。(2)化学法合成工艺以烟酰胺核糖为原料，用三氯氧磷进行磷酸化得到。虽然技术容易控制，但产品中杂质过多，分离纯化困难且总体收率很低；同时有机溶剂使用量大，对环境污染不可忽视。(3)酶作为一类与化学合成互补的、高效的生物催化剂已被广泛应用于新药研发、食品、化工等领域。目前主流的NMN生产工艺都是采用安全绿色的体外酶催化工艺。
而NMN的生物酶法制备主要有两条途径。第一条是以D-核糖和烟酰胺为起始原料，在核糖激酶、磷酸核糖焦磷酸合成酶和烟酰胺磷酸核糖转移酶等的作用下，经过三步催化反应得到NMN。该条路线底物转化率不高，且中间产物较多，后续分离纯化较困难，因而总体收率偏低，导致生产成本高。第二条路线是以烟酰胺核糖(nicotinamide riboside，NR)为起始原料，在烟酰胺核糖激酶(NR kinase，NrK)和ATP的作用下，一步反应得到NMN，收率高，产品纯度高，未来将成为NMN的主流生产方法。
如中国专利CN110373398A公开了一种烟酰胺核糖激酶突变体及其应用，该突变体的氨基酸序列与氨基酸序列SEQ ID NO.2相比，在氨基酸序列SEQ ID NO.2中第D45位、第D58位、第R161位、第Y164位进行单突变、两两联合突变、三个联合突变或四个联合突变中的一种突变；该新型烟酰胺核糖激酶突变体工业酶用于β-烟酰胺单核苷酸的合成制备，具有酶成本低，转化时间短、工艺操作简单等特点。但对于酶活并没有明显提高。
然而目前关于烟酰胺核糖激酶的研究仍然较少，限制了生物酶催化一步反应法制备NMN工业化生产中的应用。通过定向突变方法提高烟酰胺核糖激酶的酶活性，将十分有助于实现工业上减少酶量，降低生产成本。
发明内容
针对现有技术存在的不足，本发明要解决的技术问题是提供一种烟酰胺核糖激酶突变体及其编码基因和应用，以解决现有NMN制备方法原料成本较高且野生型烟酰胺核糖激酶活性不理想，难以实现工业化生产的问题。
为解决上述技术问题，本发明提供以下技术方案：
来源于Homo sapiens的野生型烟酰胺核糖激酶的氨基酸序列如SEQ ID NO:2所示，其编码基因的核苷酸序列如SEQ ID NO:1所示。
本发明针对野生型NrK，通过大分子建模技术模拟NrK的三维结构，利用能量最低原理和分子对接技术预测出可能的与催化相关的一个或多个位点。选择定点突变的位点以及突变后的氨基酸种类后，相应的引物由常州基宇生物技术有限公司合成，以NrK-pET28a(+)载体质粒为模板，PCR(Polymerase Chain Reaction，聚合酶链式反应)扩增突变DNA片段，分离纯化，然后以PCR将所得片段扩增为全长突变基因。通过将该全长突变基因克隆到pET28a(+)载体上并转入DH5a宿主菌中，经培养筛选出具有突变基因载体的阳性克隆。最后从阳性克隆中提取质粒DNA，进行DNA序列测定分析，以确定引入的突变。筛选得到的阳性质粒转入BL21(DE3)宿主菌中诱导表达，从中筛选出活性有显著提高的突变体。
具体地，本发明通过大分子建模技术预测出可能与二硫键形成相关的位点，并组合出四个两两联合突变组合，分别为S9和L102、G7和W112、F52和H146、S139和Y142。分别对这四个组合的位点进行-定点突变，利用高压液相色谱法(HPLC)来进行突变体的筛选。更为具体的为：1、当位点9的丝氨酸(S)突变为半胱氨酸(C)，位点102的亮氨酸(L)突变为半胱氨酸(C)时，突变体的催化活性相对于野生型酶来说得到了提高；2、当位点7的甘氨酸(G)突变为半胱氨酸(C)，位点112的色氨酸(W)突变为半胱氨酸(C)时，突变体单位酶活略微降低；3、当位点52的苯丙氨酸(F)突变为半胱氨酸(C)，位点146的组氨酸(H)突变为半胱氨酸(C)时，突变体酶活也得到了显著提高；4、当位点139的丝氨酸(S)突变为半胱氨酸(C)，位点142的酪氨酸(Y)突变为半胱氨酸(C)时，突变体的催化活性相对于野生型酶来说变化不大。
因此，一方面，本发明请求保护一种烟酰胺核糖激酶突变体，其氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比，在SEQ ID NO:2所示的氨基酸序列的第9位、第102位、第52位、第146位进行单突变、两两联合突变、三个联合突变或四个联合突变中的任意一种突变。
具体地，所述的烟酰胺核糖激酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比，在SEQ ID NO:2所示的氨基酸序列的第9位、第102位进行两两联合突变。
优选地，当SEQ ID NO:2所示的氨基酸序列的第9位由丝氨酸突变为半胱氨酸，第102位突变由亮氨酸突变为半胱氨酸，所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQID NO:3所示。相应地，所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ IDNO:5所示。
或，具体地，所述的烟酰胺核糖激酶突变体的氨基酸序列与氨基酸序列如SEQ IDNO:2所示的野生型烟酰胺核糖激酶相比，在SEQ ID NO:2所示的氨基酸序列的第52位、第146位进行两两联合突变。
优选地，当SEQ ID NO:2所示的氨基酸序列的第52位由苯丙氨酸突变为半胱氨酸，第146位的组氨酸突变为半胱氨酸，所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ IDNO:4所示。相应地，所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:6所示。
另一方面，本发明还请求保护上述烟酰胺核糖激酶突变体的编码基因，其核苷酸序列如SEQ ID NO:5或SEQ ID NO:6所示。
又一方面，本发明还请求保护含有上述编码基因的载体。
具体地，所述的载体可以为各种表达载体，包括但不限于pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体中的任意一种表达载体。
又一方面，本发明还请求保护含有上述编码基因的宿主细胞。
具体地，所述的宿主细胞可以为任一种合适的宿主细胞，包括但不限于大肠杆菌、枯草芽孢杆菌、链霉菌或毕赤酵母。
又一方面，本发明还请求保护上述烟酰胺核糖激酶突变体、编码基因、载体、宿主细胞在制备β-烟酰胺单核苷酸中的应用。
再一方面，本发明还提供了一种制备β-烟酰胺单核苷酸的方法，包括以下步骤：
S1.配置反应体系，包含：1-10g/L烟酰胺核糖激酶突变体，50mM pH6.0-8.0磷酸钠缓冲液，5mM ATP或ADP，10-50g/L烟酰胺核糖，50mM六偏磷酸钠，50mM七水硫酸镁；控制反应体系温度在40℃，进行搅拌反应；
S2.反应3h后进行HPLC检测；经过纯化后得β-烟酰胺单核苷酸。
反应产物经HPLC检测，反应转化率＞99％。β-烟酰胺单核苷酸的生成率可达到95％。
相对于现有技术，本发明具有以下有益效果：
本发明构建的烟酰胺核糖激酶突变体与野生型酶相比，酶活力提高了3.86-4.53倍，能显著降低酶的使用量，降低发酵容量和成本；且能极大缩短反应时间，提高NMN收率，能够满足采用生物酶法制备NMN的大规模工业化生产的需求，具有广泛的应用前景。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例中，未注明具体条件的实验方法，通常按常规条件，如《分子克隆实验指南》(J.萨姆布鲁克，D.W.拉塞尔著，黄培堂，汪嘉玺，朱厚础等译，第三版，北京：科学出版社，2002)中所述的方法进行。
实施例一原核表达体系的构建
NrK基因片段由常州基宇生物技术有限公司合成，并重组到PUC57载体上。经限制性内切酶NdeI和HindIII(购自New England Biolabs公司，NEB)在37℃双酶切4h后，1％琼脂糖凝胶电泳分离并进行切胶回收(胶回收试剂盒购自天根生化科技(北京)有限公司)。随后与同样经过双酶切的表达载体pET28a(+)(Novagen公司)，在T4 DNA连接酶(购自Takara公司)作用下置于低温连接仪里连接过夜。连接液转化DH5a感受态细胞，并进行菌落PCR筛选和测序验证，从而得到阳性重组质粒NrK-pET28a(+)。
将阳性重组质粒NrK-pET28a(+)转化表达宿主菌BL21(DE3)(购自天根生化科技(北京)有限公司)，得到原核表达菌株NrK-pET28a(+)/BL21(DE3)，作为后续定向突变和发酵的原代菌株。
用于ATP再生的多聚磷酸激酶(PPK2，来源于E.coli)由常州基宇生物技术有限公司合成，后续重组表达质粒的构建同NrK-pET28a(+)质粒的构建，转入BL21(DE3)中后得到表达菌株。
实施例二酶的摇瓶发酵制备酶冻干粉
上述构建的表达菌株NrK-pET28a(+)/BL21(DE3)、在加有终浓度为30μg/mL，硫酸卡那霉素的5mL LB液体培养基【10g/L胰蛋白胨(OXIOD)，5g/L酵母粉(OXIOD)，10g/L氯化钠(国药试剂)】中于37℃、200rpm振荡培养过夜后，按1％(V/V)比例接种于含有终浓度为30μg/mL硫酸卡那霉素的400mL LB液体培养基中，于37℃、200rpm振荡培养。待OD600在0.8-1.0之间时，加入终浓度为0.1mM的诱导剂IPTG(异丙基-β-D-硫代半乳糖苷)，并在25℃诱导过夜。菌体在4℃、8000rpm条件下离心收集，然后悬浮于50mM pH7.0磷酸钠缓冲液中，超声破碎(200W，3s/5s，30min)，4℃、12000rpm离心20min，取上清进行镍柱亲和层析纯化，经咪唑洗脱、除盐后冷冻干燥，即得酶冻干粉。
实施例三突变体的构建和筛选
突变体的构建：采用大分子建模技术预测出可能有益的突变位点为S9和L102、G7和W112、F52和H146、S139和Y142四个两两联合突变组合。随后以NrK-pET28a(+)重组质粒为模板，使用合成的相应引物，第一次PCR扩增突变DNA片段，然后以PCR将所得片段作为模板，第二次PCR扩增全长得到NrK的突变基因。
其中：
9位点突变
正向引物(SEQ ID NO:7)：5'CCCATATGAAAACCTTCATCATTGGTATTTGCGG 3'，
反向引物为T7ter通用引物(SEQ ID NO:8)：5'TGCTAGTTATTGCTCAGCGG 3'；
102位点突变
正向引物(SEQ ID NO:9)：5'GAAGGTTTTCTGTGCTTCAACTACA 3'，
反向引物(SEQ ID NO:10)：5'TGTAGTTGAAGCACAGAAAACCTTC 3'；
7位点突变
正向引物(SEQ ID NO:11)：5'CCCATATGAAAACCTTCATCATTTGCATTAGCGG 3'，
反向引物为T7ter通用引物(SEQ ID NO:12)：5'TGCTAGTTATTGCTCAGCGG 3'；
112位点突变
正向引物(SEQ ID NO:13)：5'GGACACCATTTGCAATCGCAGCTAT 3'，
反向引物(SEQ ID NO:14)：5'ATAGCTGCGATTGCAAATGGTGTCC 3'；
52位点突变
正向引物(SEQ ID NO:15)：5'TAAAAACGGCTGCCTGCAATATGAC 3'，
反向引物(SEQ ID NO:16)：5'GTCATATTGCAGGCAGCCGTTTTTA 3'；
146位点突变
正向引物(SEQ ID NO:17)：5'TACTTCGACGGTTGCGTCTGGCCGAT 3'，
反向引物(SEQ ID NO:18)：5'ATCGGCCAGACGCAACCGTCGAAGTA 3'；
139和142位点突变
正向引物(SEQ ID NO:19)：5'AACCGCCGGATTGTCCGGGCTGCTTCGACGGT 3'，
反向引物(SEQ ID NO:20)：5'ACCGTCGAAGCAGCCCGGACAATCCGGCGGTT 3'；
突变体培养：将上述突变得到的质粒转化BL21(DE3)宿主菌后，涂布于含30μg/mL卡那霉素的LB固体培养基上，37℃倒置培养过夜，随后从平板上挑取单克隆，置于含有30μg/mL硫酸卡那霉素的5mL LB液体培养基中进行培养。过夜培养的菌液再按1％(V/V)比例接种于含有30μg/mL硫酸卡那霉素的100mL LB液体培养基中，37℃、200rpm振荡培养4h后加入终浓度为0.1mM的IPTG进行诱导，25℃培养过夜。4℃、8000rpm离心10min收集菌体，用50mMpH7.0磷酸钠缓冲液悬浮后超声破碎(200W，3s/5s，30min)，4℃、12000rpm离心20min，取上清进行单位酶活测定。
突变体的筛选：底物NR浓度10g/L，ATP 30mM，10mM七水硫酸镁，加入适量上述制备的上清液，用10mM pH7.0磷酸钠缓冲液补充体积至2mL，置于37℃恒温磁力搅拌器下搅拌反应。反应20min后取样进行HPLC检测。
实验结果显示，突变体酶活得到显著提高的克隆中含有的突变位点组合如下：
位点9的丝氨酸(S)突变为半胱氨酸(C)和位点102的亮氨酸(L)突变为半胱氨酸(C)；位点52的苯丙氨酸(F)突变为半胱氨酸(C)和位点146的组氨酸(H)突变为半胱氨酸(C)。
具体酶活数值见下表1。
表1不同突变组合的酶活
| 突变体 | 单位酶活 | 提高倍数 |
| Nrk | 1.83U/mg | -- |
| hNrK-G7C-W112C | 1.37U/mg | 0.74 |
| hNrK-F52C-H146C | 8.3U/mg | 4.53 |
| hNrK-S9C-L102C | 7.06U/mg | 3.86 |
| hNrK-S139C-Y142C | 1.75U/mg | 0.96 |
*1U定义为单位时间内(1min)生产1μmol产物NMN所需的酶量。
因此，当SEQ ID NO:2所示的氨基酸序列的第9位由丝氨酸突变为半胱氨酸，第102位突变由亮氨酸突变为半胱氨酸，烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:3所示。相应地，所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示。
当SEQ ID NO:2所示的氨基酸序列的第52位由苯丙氨酸突变为半胱氨酸，第146位的组氨酸突变为半胱氨酸，所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:4所示。相应地，所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:6所示。
实施例四突变体的生物催化
将160mg底物NR溶解于1.6mL 50mM pH6.5磷酸钠缓冲液中，待底物完全溶解后加入50mM六偏磷酸钠、5mM ATP、50mM七水硫酸镁、烟酰胺核糖激酶突变体hNrK-F52C-H146C和PPK2酶量分别是2g/L和1g/L，在40℃恒温磁力搅拌器下搅拌反应，反应3h后进行HPLC检测；经过纯化后得β-烟酰胺单核苷酸。底物转化率大于99％，β-烟酰胺单核苷酸的生成率可达到95％。
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。
序列表
<110> 中山俊凯生物技术开发有限公司
<120> 一种烟酰胺核糖激酶突变体及其编码基因和应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 600
<212> DNA
<213> 人(Homo sapiens)
<400> 1
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ccggaaagcg aaatcgaaac cgataaaaac ggctttctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggtcacgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 2
<211> 199
<212> PRT
<213> 人(Homo sapiens)
<400> 2
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly His Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 3
<211> 199
<212> PRT
<213> 人(Homo sapiens)
<400> 3
Met Lys Thr Phe Ile Ile Gly Ile Cys Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Phe Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Cys Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly His Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 4
<211> 199
<212> PRT
<213> 人(Homo sapiens)
<400> 4
Met Lys Thr Phe Ile Ile Gly Ile Ser Gly Val Thr Asn Ser Gly Lys
1 5 10 15
Thr Thr Leu Ala Lys Asn Leu Gln Lys His Leu Pro Asn Cys Ser Val
20 25 30
Ile Ser Gln Asp Asp Phe Phe Lys Pro Glu Ser Glu Ile Glu Thr Asp
35 40 45
Lys Asn Gly Cys Leu Gln Tyr Asp Val Leu Glu Ala Leu Asn Met Glu
50 55 60
Lys Met Met Ser Ala Ile Ser Cys Trp Met Glu Ser Ala Arg His Ser
65 70 75 80
Val Val Ser Thr Asp Gln Glu Ser Ala Glu Glu Ile Pro Ile Leu Ile
85 90 95
Ile Glu Gly Phe Leu Leu Phe Asn Tyr Lys Pro Leu Asp Thr Ile Trp
100 105 110
Asn Arg Ser Tyr Phe Leu Thr Ile Pro Tyr Glu Glu Cys Lys Arg Arg
115 120 125
Arg Ser Thr Arg Val Tyr Gln Pro Pro Asp Ser Pro Gly Tyr Phe Asp
130 135 140
Gly Cys Val Trp Pro Met Tyr Leu Lys Tyr Arg Gln Glu Met Gln Asp
145 150 155 160
Ile Thr Trp Glu Val Val Tyr Leu Asp Gly Thr Lys Ser Glu Glu Asp
165 170 175
Leu Phe Leu Gln Val Tyr Glu Asp Leu Ile Gln Glu Leu Ala Lys Gln
180 185 190
Lys Cys Leu Gln Val Thr Ala
195
<210> 5
<211> 600
<212> DNA
<213> 人(Homo sapiens)
<400> 5
atgaaaacct tcatcattgg tatttgcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctttctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgtgcttca actacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggtcacgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 6
<211> 600
<212> DNA
<213> 人(Homo sapiens)
<400> 6
atgaaaacct tcatcattgg tattagcggt gtgacgaaca gcggtaaaac gaccctggcg 60
aaaaacctgc aaaaacatct gccgaattgt agcgtgattt ctcaggatga ctttttcaaa 120
ccggaaagcg aaatcgaaac cgataaaaac ggctgcctgc aatatgacgt tctggaagca 180
ctgaatatgg aaaaaatgat gagtgcgatt tcctgctgga tggaaagtgc ccgccattca 240
gtggtttcga cggatcagga atccgcggaa gaaattccga tcctgattat cgaaggtttt 300
ctgctgttca actacaaacc gctggacacc atttggaatc gcagctattt tctgacgatc 360
ccgtacgaag aatgtaaacg tcgccgtagc acccgtgttt atcaaccgcc ggattctccg 420
ggctacttcg acggttgcgt ctggccgatg tatctgaaat accgtcagga aatgcaagat 480
atcacctggg aagtcgtgta tctggatggc acgaaatctg aagaagacct gttcctgcaa 540
gtgtatgaag acctgatcca agaactggcg aaacaaaaat gcctgcaagt gaccgcataa 600
<210> 7
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cccatatgaa aaccttcatc attggtattt gcgg 34
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgctagttat tgctcagcgg 20
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gaaggttttc tgtgcttcaa ctaca 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tgtagttgaa gcacagaaaa ccttc 25
<210> 11
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cccatatgaa aaccttcatc atttgcatta gcgg 34
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgctagttat tgctcagcgg 20
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggacaccatt tgcaatcgca gctat 25
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atagctgcga ttgcaaatgg tgtcc 25
<210> 15
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
taaaaacggc tgcctgcaat atgac 25
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gtcatattgc aggcagccgt tttta 25
<210> 17
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tacttcgacg gttgcgtctg gccgat 26
<210> 18
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atcggccaga cgcaaccgtc gaagta 26
<210> 19
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aaccgccgga ttgtccgggc tgcttcgacg gt 32
<210> 20
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
accgtcgaag cagcccggac aatccggcgg tt 32
Claims (10)
1.一种烟酰胺核糖激酶突变体，其特征在于，所述的烟酰胺核糖激酶突变体的氨基酸序列与氨基酸序列如SEQ ID NO:2所示的野生型烟酰胺核糖激酶相比，在SEQ ID NO:2所示的氨基酸序列的第9位、第102位、第52位、第146位进行单突变、两两联合突变、三个联合突变或四个联合突变中的任意一种突变。
2.根据权利要求1所述的烟酰胺核糖激酶突变体，其特征在于，当SEQ ID NO:2所示的氨基酸序列的第9位由丝氨酸突变为半胱氨酸，第102位突变由亮氨酸突变为半胱氨酸，所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:3所示。
3.根据权利要求1所述的烟酰胺核糖激酶突变体，其特征在于，当SEQ ID NO:2所示的氨基酸序列的第52位由苯丙氨酸突变为半胱氨酸，第146位的组氨酸突变为半胱氨酸，所述的烟酰胺核糖激酶突变体的氨基酸序列如SEQ ID NO:4所示。
4.权利要求2所述的烟酰胺核糖激酶突变体的编码基因，其特征在于，所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:5所示。
5.权利要求3所述的烟酰胺核糖激酶突变体的编码基因，其特征在于，所述的烟酰胺核糖激酶突变体的编码基因的核苷酸序列如SEQ ID NO:6所示。
6.含有权利要求4或5所述的编码基因的载体。
7.根据权利要求6所述的载体，其特征在于，所述的载体为pET表达载体、pCW表达载体、pUC表达载体或pPIC9k表达载体。
8.含有权利要求4或5所述的编码基因的宿主细胞，其特征在于，所述的宿主细胞为大肠杆菌、枯草芽孢杆菌、链霉菌或毕赤酵母。
9.权利要求1-3任一项所述的烟酰胺核糖激酶突变体、权利要求4或5所述的编码基因、权利要求6或7所述的载体、权利要求8所述的宿主细胞在制备β-烟酰胺单核苷酸中的应用。
10.一种制备β-烟酰胺单核苷酸的方法，其特征在于，所述的方法包括以下步骤：
S1.配置反应体系，包含：1-10g/L权利要求1-3任一项所述的烟酰胺核糖激酶突变体，50mM pH6.0-8.0磷酸钠缓冲液，5mM ATP或ADP，10-50g/L烟酰胺核糖，50mM六偏磷酸钠，50mM七水硫酸镁；控制反应体系温度在40℃，进行搅拌反应；
S2.反应3h后进行HPLC检测；经过纯化后得β-烟酰胺单核苷酸。
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| CN113621595A (zh) * | 2021-04-30 | 2021-11-09 | 杭州灵犀健康科技有限公司 | 一种工程化核糖激酶、其编码基因、表达载体、工程菌及其应用 |
| CN113832125A (zh) * | 2021-10-19 | 2021-12-24 | 中山百灵生物技术股份有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
| CN114044800A (zh) * | 2015-08-26 | 2022-02-15 | 艾其林医药公司 | 用于治疗医学障碍的芳基、杂芳基和杂环化合物 |
| CN114107160A (zh) * | 2021-12-27 | 2022-03-01 | 浙江工业大学 | 一种烟酰胺核糖激酶基因工程菌及其应用 |
| CN114606213A (zh) * | 2022-01-28 | 2022-06-10 | 浙江工业大学 | 一种多聚磷酸激酶突变体、工程菌及其应用 |
| CN114990088A (zh) * | 2022-06-24 | 2022-09-02 | 中食都庆（山东）生物技术有限公司 | 烟酰胺核糖激酶突变体及其重组菌与nmn的制备方法 |
| CN115044565A (zh) * | 2022-06-08 | 2022-09-13 | 中山俊凯生物技术开发有限公司 | 一种胆绿素还原酶突变体及其编码基因和应用 |
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| CN114044800A (zh) * | 2015-08-26 | 2022-02-15 | 艾其林医药公司 | 用于治疗医学障碍的芳基、杂芳基和杂环化合物 |
| CN113106080B (zh) * | 2021-03-31 | 2022-02-25 | 深圳希吉亚生物技术有限公司 | 烟酰胺磷酸核糖转移酶突变体及其应用 |
| CN113106080A (zh) * | 2021-03-31 | 2021-07-13 | 深圳希吉亚生物技术有限公司 | 烟酰胺磷酸核糖转移酶突变体及其应用 |
| CN113621595A (zh) * | 2021-04-30 | 2021-11-09 | 杭州灵犀健康科技有限公司 | 一种工程化核糖激酶、其编码基因、表达载体、工程菌及其应用 |
| CN113265382A (zh) * | 2021-06-24 | 2021-08-17 | 洛阳华荣生物技术有限公司 | 多聚磷酸激酶突变体 |
| CN113265382B (zh) * | 2021-06-24 | 2023-11-10 | 洛阳华荣生物技术有限公司 | 多聚磷酸激酶突变体 |
| CN113832125B (zh) * | 2021-10-19 | 2023-09-26 | 中山百灵生物技术股份有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
| CN113832125A (zh) * | 2021-10-19 | 2021-12-24 | 中山百灵生物技术股份有限公司 | 一种烟酰胺核糖激酶突变体及其编码基因和应用 |
| CN114107160A (zh) * | 2021-12-27 | 2022-03-01 | 浙江工业大学 | 一种烟酰胺核糖激酶基因工程菌及其应用 |
| CN114606213A (zh) * | 2022-01-28 | 2022-06-10 | 浙江工业大学 | 一种多聚磷酸激酶突变体、工程菌及其应用 |
| CN114606213B (zh) * | 2022-01-28 | 2024-05-03 | 浙江工业大学 | 一种多聚磷酸激酶突变体、工程菌及其应用 |
| CN115044565A (zh) * | 2022-06-08 | 2022-09-13 | 中山俊凯生物技术开发有限公司 | 一种胆绿素还原酶突变体及其编码基因和应用 |
| CN114990088A (zh) * | 2022-06-24 | 2022-09-02 | 中食都庆（山东）生物技术有限公司 | 烟酰胺核糖激酶突变体及其重组菌与nmn的制备方法 |
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