CN114277013A - 一种nad激酶突变体及其应用 - Google Patents
一种nad激酶突变体及其应用 Download PDFInfo
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- CN114277013A CN114277013A CN202011031687.7A CN202011031687A CN114277013A CN 114277013 A CN114277013 A CN 114277013A CN 202011031687 A CN202011031687 A CN 202011031687A CN 114277013 A CN114277013 A CN 114277013A
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Abstract
本发明公开了一种NAD激酶突变体，该NAD激酶突变体能够将NAD转化为NADP。相对于野生型NAD激酶，该NAD激酶突变体提高了催化效能，转化率有大幅度提高，降低了底物残留，方便了后处理的纯化工艺，降低了生产成本，更适于工业化生产应用。
Description
技术领域：
本发明属于蛋白质工程技术领域，具体涉及一种能够将NAD转化为NADP的NAD激酶突变体。
背景技术：
氧化型β-烟酰胺腺嘌呤二核苷酸磷酸(简称：辅酶Ⅱ，英文名：Nicotinamideadenine dinucleotide phosphate，NADP)，是一种极为重要的核苷酸类辅酶。氧化型辅酶II在氧化还原反应中传递质子、电子及能量，并且参与到很多细胞代谢反应中。辅酶II在生命科学、酶催化不对称合成、医疗保健领域具有广泛的应用。
辅酶II在生物中广泛存在，但含量极低。目前合成NADP的方法主要分为化学法和生物法。其中，化学法以烟酰胺为原料，经多步反应合成出NADP，但化学法存在反应路线长、反应条件苛刻、选择性差、容易生成副产物、收率低等问题，而且生产成本较高，需要使用有机溶剂，会造成环境污染问题。
生物法又分为传统的发酵法和酶法，其中发酵法是采用发酵或其它微生物培养技术，通过对酵母或其它微生物的分离、提取，得到NADP，比如罗氏公司就是使用发酵法制备NADP。但是该路线的原料耗费、能源消耗都非常大，原子利用率低、产量有限，生产成本高，限制了NADP的广泛应用。而酶催化合成NADP是一种更为高效的反应，具有反应条件温和、立体选择性强、相比于发酵法转化率高等优点。
目前，天然微生物来源的NAD激酶存在着转化率不彻底的问题，而且由于NAD和NADP结构类似，反应结束后存在显著的底物残留，需要后处理才能够去除未完全转化的NAD，因此使得后处理工艺变复杂，工业化生产成本居高不下。
因此，获得具有更高转化率的NAD激酶是降低NADP的生物催化合成成本、提高NAD激酶的工业应用价值、促进生物催化技术在NADP产业化生产中应用的关键因素。
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NAD激酶突变体。
一方面，本发明提供的NAD激酶突变体的氨基酸序列是以SEQ ID NO.2所示的NAD激酶为参考序列发生突变的氨基酸序列，突变位点为第127位的甲硫氨酸突变为丝氨酸，第182位的苯丙氨酸突变为丙氨酸，第211位的谷氨酰胺突变为组氨酸。
进一步，所述NAD激酶突变体的氨基酸序列如SEQ ID NO.4所示。
进一步，所述NAD激酶突变体的核苷酸序列如SEQ ID NO.3所示。
进一步，所述NAD激酶突变体的野生型基因序列来源于闪烁古生球菌(Archaeoglobus fulgidus)，野生型模板NCBI的登录号为WP_048096396.1。
更进一步，NAD激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NAD激酶突变体可以将NAD在ATP存在下转化为NADP。
进一步，所述NAD激酶突变体为NAD激酶酶粉或含有该NAD激酶的全细胞或细胞破碎液。
进一步，所述NAD激酶酶粉浓度为1～10g/L。
进一步，所述NAD激酶细胞浓度为5～50g/L。
进一步，所述NAD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NAD激酶突变体能够将NAD转化为NADP，转化率能够从80％提高至99％，提高了催化效能，降低了底物残留，方便了后处理的纯化工艺，降低了生产成本，具有重大的工业化应用价值。
附图说明
无
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NAD激酶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NAD激酶通过计算机模拟结构，与底物进行对接，推测活性中心附近的127位点、182位点、211位点在与催化作用密切相关，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。
按照表2配制转化体系，用排枪吸取600uL转入上述离心后的96孔板中，在42℃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 |
| NAD | 20g/L |
| ATP | 20g/L |
| MgCl<sub>2</sub> | 10mM |
| pH7.0磷酸钾Buffer | 100mM |
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，选取转化率最高(大于WT)的突变体进行测序，根据序列比对发现突变体分别为F182A,M128S,Q211H。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 | M128 | 2C9 | ATG->AGT | M128S |
| Mu02 | F182 | 1D10 | TTC->GCG | F182A |
| Mu03 | Q211 | 1F4 | CAG->CAT | Q211H |
实施例4组合突变体的构建
在Q211H突变体(Mu03)的基础上，对M128S、F182A设计定点突变引物，顺次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组合突变体的反应测试
按照表4的体系配制反应体系，反应体积为10mL。所有物料加完后，在42℃的恒温振荡器中，300rpm的转速下反应18小时，取样HPLC检测，结果显示，Mu04在18小时后几乎可转化完底物，转化率99.1％，而WT对照组转化率为80.7％。
表4比较突变体催化效果的反应液体系
| 原料 | 浓度 |
| NAD | 20g/L |
| ATP | 20g/L |
| MgCl<sub>2</sub> | 10mM |
| pH7.0磷酸钾Buffer | 100mM |
| 细胞 | 50g/L |
实施例6 NAD激酶Mu04突变体细胞/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。离心收集菌体得到Mu04突变体细胞。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NAD激酶Mu04突变体的酶粉。
实施例7 NADP的制备
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 MgCl2，2g NAD，2g ATP，用水补足100mL，加入Mu04细胞5g，42℃水浴锅中，搅拌反应18小时。取样HPLC检测，结果显示转化率99.2％。
实施例8 NADP的制备
向1L圆底烧瓶中加入500mL事先配好的pH 7.0的200mM的磷酸盐缓冲液、10mL事先配好的1M MgCl2，20g NAD，20g ATP，用水补足1L体积，加入Mu04酶粉10g，42℃水浴锅中，搅拌反应18小时。取样HPLC检测，结果显示转化率99.1％。
序列表
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Claims (5)
1.一种NAD激酶突变体，其特征在于，以SEQ ID NO.2所示的野生型NAD激酶的氨基酸序列为参考序列，第127位的甲硫氨酸突变为丝氨酸，第182位的苯丙氨酸突变为丙氨酸，第211位的谷氨酰胺突变为组氨酸。
2.如权利要求1所述的NAD激酶突变体，其特征在于，所述NAD激酶突变体氨基酸序列如SEQ ID NO.4所示。
3.如权利要求1所述的NAD激酶突变体，其特征在于，所述NAD激酶突变体的基因核苷酸序列如SEQ ID NO.3所示。
4.如权利要求1所述的NAD激酶突变体，其特征在于，所述NAD激酶突变体表达于基因工程菌。
5.如权利要求1所述的NAD激酶突变体，其特征在于，所述NAD激酶突变体能够将NAD转化为NADP。
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