CN114438052B - 一种烟酰胺单核苷酸腺苷转移酶突变体及其应用 - Google Patents
一种烟酰胺单核苷酸腺苷转移酶突变体及其应用 Download PDFInfo
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- CN114438052B CN114438052B CN202011191122.5A CN202011191122A CN114438052B CN 114438052 B CN114438052 B CN 114438052B CN 202011191122 A CN202011191122 A CN 202011191122A CN 114438052 B CN114438052 B CN 114438052B
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Abstract
本发明公开了一种烟酰胺单核苷酸腺苷转移酶突变体，能够将NMN转化为NAD。该酶的热稳定性好，制备成为固定化细胞后，可以反复套用20次以上，大大降低了NAD的生产成本，有利于提高产品的市场竞争力，具有实际的工业化应用价值。
Description
技术领域：
本发明属于蛋白质工程技术领域，具体涉及一种烟酰胺单核苷酸腺苷转移酶突变体及其能够将NMN转化为NAD。
背景技术：
烟酰胺腺嘌呤二核苷酸(简称：辅酶I，英文名：nicotinamide adeninedinucleotide，NAD)是生物体内不可缺少的小分子化合物，参与氧化还原反应，能量传递，物质代谢和信号传导等过程。NAD是生物体内非常常见的一种脱氢酶的辅酶，脱氢酶在生物体的新陈代谢中起到了决定性的作用，一些生物体基本的代谢运动，比如蛋白质分解，碳水化合物分解，脂肪分解，都离不开脱氢酶。由于脱氢酶必须在NAD的参与下才能促使代谢运作，所以是NAD生物体代谢中不可缺少的物质。
NAD是现代生物催化反应必不可少的辅酶之一。现代的生物催化技术模拟了生物体内利用酶来完成的反应，这种反应的模拟条件是严格根据生物体内的代谢特征来进行的，所以像常见的亮氨酸脱氢酶、甲酸铵脱氢酶、葡萄糖脱氢酶、部分酮还原酶催化的手性还原都需要NAD的帮助来完成整个反应。而在材料、化工、食品、制药工业中，氧化还原反应(特别是手性还原)是数目最庞大、研究最深入、工艺最成熟的一类反应，所以工业领域对NAD的需求是巨大的。
关于NAD的制备方法的报道很多，主要有化学合成、酵母提取和酶法制备三类。
化学合成法由于转化率低，所用试剂昂贵，且有大量的过硅胶柱和凝胶色谱柱的操作，在工业生产上受到一定的限制。
从酵母中提取大方法虽然工艺成熟，但设备庞大，浓缩耗能高，生产效率低，成本高。
酶法合成主要是使用β-烟酰胺单核苷酸(NMN)和ATP作为底物，在烟酰胺单核苷酸腺苷转移酶(nicotinamide mononucleotide adenylyltransferase，NMNAT)的作用下，将NMN腺苷化，合成NAD。由于生产中需要使用大量的酶，占据了大量的生产成本。为了降低生产成本，可以将酶制备成固定化酶来增加酶的反复套用次数，但酶本身热稳定性的高低制约了固定化技术能否顺利实施。如果酶本身在反应温度下就会逐渐失活，虽然可能在第一次反应中对转化率的影响还不是很明显，但后续批次的套用效果会迅速地变差。
因此，获得具有较高热稳定性的烟酰胺单核苷酸腺苷转移酶对于后续的制备固定化酶和反复套用具有直接的正面影响。
发明内容：
本发明的目的在于针对现有技术的不足，提供一种新的烟酰胺单核苷酸腺苷转移酶突变体。
一方面，本发明提供的烟酰胺单核苷酸腺苷转移酶突变体的氨基酸序列是以SEQID NO.2所示的烟酰胺单核苷酸腺苷转移酶为参考序列发生突变的氨基酸序列，突变位点为第32位的天冬氨酸突变为苏氨酸，第133位的精氨酸突变为甘氨酸，第166位的甘氨酸突变为脯氨酸。
进一步，所述烟酰胺单核苷酸腺苷转移酶突变体的氨基酸序列如SEQ ID NO.4所示。
进一步，所述烟酰胺单核苷酸腺苷转移酶突变体的核苷酸序列如SEQ ID NO.3所示。
进一步，所述烟酰胺单核苷酸腺苷转移酶突变体的野生型基因序列来源于金黄色葡萄球菌(Staphylococcus aureus)，野生型模板NCBI的登录号为WP_031923054.1。
更进一步，烟酰胺单核苷酸腺苷转移酶突变体表达于基因工程菌，优选大肠杆菌或酵母菌。
另一方面，本发明提供的烟酰胺单核苷酸腺苷转移酶突变体可以将NMN转化为NAD。
进一步，所述烟酰胺单核苷酸腺苷转移酶突变体为烟酰胺单核苷酸腺苷转移酶酶粉或含有该烟酰胺单核苷酸腺苷转移酶的全细胞或细胞破碎液。
进一步，所述烟酰胺单核苷酸腺苷转移酶酶粉浓度为1～10g/L。
进一步，所述烟酰胺单核苷酸腺苷转移酶细胞浓度为5～50g/L。
进一步，所述NMN浓度为2～20g/L。
进一步，该反应在缓冲溶液中进行，其中缓冲溶液为磷酸盐缓冲液或三乙醇胺缓冲液，优选磷酸盐缓冲液，缓冲液的浓度为50～100mmol/L。
进一步，该反应在pH＝5～8、温度为20～45℃下进行。
本发明的有益效果在于，本发明公开的烟酰胺单核苷酸腺苷转移酶突变体能够将NMN转化为NAD。该酶的热稳定性好，制备成为固定化细胞后，可以反复套用20次以上，大大降低了NAD的生产成本，有利于提高产品的市场竞争力，具有实际的工业化应用价值。
具体实施方式
下面结合具体实施例对本发明的技术内容作进一步的阐述，其目的是为了更好的理解本发明的内容，但本发明的保护范围不限于此。
实施例1构建烟酰胺单核苷酸腺苷转移酶突变体的定点饱和突变体库
对SEQ ID No.2(对应的核苷酸序列为SEQ ID No.1)的烟酰胺单核苷酸腺苷转移酶突变体通过计算机模拟结构，与底物进行对接，推测位于loop区域的32位点、133位点、166位点可能与酶的稳定性作用相关，对这些位点构建饱和突变体库，具体序列信息见表1。
表1饱和突变的引物序列
表1中带下划线的序列为突变位点，利用全质粒PCR扩增反应，扩增出带有突变基因的载体。接着利用DpnI限制性内切酶对PCR产物进行重组质粒模板消化，再经过纯化之后，转化入大肠杆菌BL21(DE3)感受态，之后将其涂布于含有50ug/L Kan的LB平板上，在37℃的培养箱中倒置培养18小时长出单克隆。
实施例2阳性克隆的高通量筛选
对每个突变体随机挑选188个单克隆进行96孔板振荡培养，每个96孔板接种两个未突变的菌种为对照组，共计6块96孔板。具体操作为：在无菌的96孔板中加入400uL的LB培养基，37℃培养16小时，按照10％的接种量，转接于含有2YT培养基的第二个96孔板中培养，培养基中加入抗生素和终浓度为0.1mM的IPTG，于25℃诱导表达24小时。培养结束后离心弃上清，冻存于-20℃冰箱中待用。
筛选时，将96孔板从冰箱中取出，每孔加入300uL 10mM PBS(pH 7.0)，于50℃烘箱热失活6小时。按照表2配制测活体系，用排枪吸取300uL转入上述热失活后的96孔板中，在30℃的恒温振荡器中，300rpm的转速下反应18小时。然后对所有转化反应液进行HPLC检测，选取转化率高于对照组的作为候选，再复测确认。
表2筛选饱和突变体库的反应液体系
| 原料 | 浓度 |
| NMN | 100mM |
| ATP | 100mM |
| MgCl2 | 20mM |
| pH7.0磷酸钾Buffer | 200mM |
实施例3阳性候选突变体的测序
在每个饱和突变体文库中，选取热失活后转化率最高(大于对照组WT)的突变体进行测序，根据序列比对发现突变体分别为D32T，R133G，G166P。表3为测序获得的序列突变信息，将其命名为Mu01-Mu03。
表3测序后的密码子和氨基酸突变信息
| Name | Site | Sample | Codon | Mutation |
| Mu01 | D32 | 1F9 | GAT->ACT | D32T |
| Mu02 | R133 | 1D6 | CGA->GGT | R133G |
| Mu03 | G166 | 2H3 | GGG->CCG | G166P |
实施例4组合突变体的构建
在D32T突变体(Mu01)的基础上，对R133G，G166P设计定点突变引物，构建三个位点的组合突变体，即Seq ID NO.4所示氨基酸序列，将其命名为Mu04。对Mu04和未突变的对照组WT同时接种5mL含卡那霉素的LB试管培养基，37℃培养12小时，按1％接种量转接活化培养物至100mL含卡那霉素的2YT液体培养基中，37℃培养OD至0.6～0.8，加入IPTG(终浓度0.1mM)于25℃诱导培养16小时，离心收集菌体。
实施例5组合突变体的热稳定性测试
将组合突变体和未突变的野生型菌体分别配成100g/L细胞悬液300uL，置于50℃烘箱热失活6小时。按照表2配制测活体系，用排枪吸取300uL转入上述热失活后的96孔板中，在30℃的恒温振荡器中，300rpm的转速下反应18小时。然后对反应液进行HPLC检测，结果显示，Mu04在热失活6小时后，反应18小时后的底物转化率为97.1％，而WT对照组转化率为30.7％。
实施例6烟酰胺单核苷酸腺苷转移酶突变体Mu04细胞/酶粉的制备
将Mu04突变体接种至5mL含卡那霉素的LB试管培养基中活化培养(37℃培养12小时)，按1％接种量转接活化培养物至400mL含卡那霉素的2YT液体培养基中，37℃培养OD至0.6～0.8，加入IPTG(终浓度0.1mM)于25℃诱导培养16小时，离心收集菌体即得到烟酰胺单核苷酸腺苷转移酶突变体Mu04的菌体细胞。
用40mL磷酸盐缓冲液(10mM，pH 7.5)重悬20g菌体后，于均质机中均质破碎，离心收集上清，-20℃预冻后真空冷冻干燥48小时后碾碎，即得烟酰胺单核苷酸腺苷转移酶突变体Mu04的酶粉。
实施例7烟酰胺单核苷酸腺苷转移酶固定化细胞的制备
将Mu04的菌体细胞加入到100mM磷酸盐缓冲液中，制成浓度为20％(v/v)的菌悬液。向菌悬液中添加氨基型树脂载体，搅拌均匀，再加入戊二醛进行交联，25℃搅拌6小时后，过滤抽干，再用4℃的蒸馏水淋洗固定化细胞两次，真空抽干即可获得固定化Mu04细胞。
实施例8用Mu04细胞制备NAD
向200mL圆底烧瓶中加入50mL事先配好的pH 7.0的200mM的磷酸盐缓冲液、1mL事先配好的1M MgCl2，1.7g NMN，3.0g ATP，调pH 7.0，用水补足100mL，加入Mu04细胞3g，30℃水浴锅中，搅拌反应18小时。取样HPLC检测，结果显示转化率为99.2％。
实施例9用固定化Mu04细胞制备NAD
向200mL圆底烧瓶中加入50mL事先配好的pH 7.0的200mM的磷酸盐缓冲液、1mL事先配好的1M MgCl2，1.7g NMN，3.0g ATP，调pH 7.0，用水补足100mL，加入Mu04固定化细胞6.5g，30℃水浴锅中，搅拌反应18小时。取样HPLC检测，结果显示转化率99.0％。
离心收集固定化细胞，继续按上述体系进行反复套用，第20次套用时，转化率95.2％。
序列表
<120> 一种烟酰胺单核苷酸腺苷转移酶突变体及其应用
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<170> SIPOSequenceListing 1.0
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Ile Ala Ile Gln Ile Pro Arg Val Asp Ile Ser Ser Thr Met Ile Arg
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Claims (4)
1.一种烟酰胺单核苷酸腺苷转移酶突变体，其特征在于，所述烟酰胺单核苷酸腺苷转移酶突变体氨基酸序列如SEQ ID NO.4所示。
2.如权利要求1所述的烟酰胺单核苷酸腺苷转移酶突变体，其特征在于，编码所述烟酰胺单核苷酸腺苷转移酶突变体的基因的核苷酸序列如SEQ ID NO.3所示。
3.如权利要求1所述的烟酰胺单核苷酸腺苷转移酶突变体，其特征在于，所述烟酰胺单核苷酸腺苷转移酶突变体表达于基因工程菌。
4.如权利要求1所述的烟酰胺单核苷酸腺苷转移酶突变体，其特征在于，所述烟酰胺单核苷酸腺苷转移酶突变体能够将NMN转化为NAD。
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