CN116064443A - 亚胺还原酶突变体及其应用 - Google Patents
亚胺还原酶突变体及其应用 Download PDFInfo
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- CN116064443A CN116064443A CN202111270046.1A CN202111270046A CN116064443A CN 116064443 A CN116064443 A CN 116064443A CN 202111270046 A CN202111270046 A CN 202111270046A CN 116064443 A CN116064443 A CN 116064443A
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Abstract
本发明提供了一种亚胺还原酶突变体及其在(S)‑去甲烟碱合成中的应用，具体的，本发明对来源于Myxococcus fulvus的亚胺还原酶MfIR(WP_074958336.1)进行定向进化，获得酶活力及底物耐受提高的亚胺还原酶突变体，利用得到的突变体还原麦斯明从而制备高手性纯度的(S)‑去甲烟碱的方法，所述的Myxococcusfulvus来源的亚胺还原酶突变体活性高，反应底物浓度、反应产率以及产物的光学纯度高，反应过程中操作简单，能耗低，符合绿色化学要求，可应用于工业生产(S)‑去甲烟碱化合物的生物转化制备。
Description
技术领域
本发明属于生物催化领域，涉及对一种利用来源于粘球菌属的亚胺还原酶进行突变，并利用突变体为生物催化剂，以NADP(H)为辅酶，还原麦斯明(CAS:532-12-7)生成(S)-去甲烟碱的方法，产物光学纯度>99％。
背景技术
(S)-去甲烟碱，又名(S)-降烟碱，英文名(S)-(-)-Nornicotine，CAS号：494-97-3，分子式C9H12N2，结构式如下：
(S)-去甲烟碱用于合成尼古丁疫苗中的尼古丁半抗原的前期研究以及治疗尼古丁上瘾的前期研究中，也是治疗帕金森氏病、阿尔茨海默氏病和图雷特综合征方面的潜在的有益化合物前体[Viswanath A,Joseph L,[J]ACS Comb.Sci.2017,19,286-298]。
(S)-去甲烟的合成方法主要包括化学法合成或生物酶催化法合成，使用化学法合成(S)-去甲烟碱需要多步反应，过程中会用到手性衍生试剂或者金属催化剂等才能完成，条件苛刻，易造成环境污染，得到的产品光学纯度不高，难以达到98.0％以上，收率较低，在实际大规模生产中有诸多限制[Charles H.M.,Steven J.Q.[J]Journal of MedicinalChemistry,2017,19,286-298]。因此，探索更加绿色高效的生物酶催化法显得尤为重要。
生物催化合成(S)-去甲烟的报道较少，2021年，Raymond McCague等利用来源的亚胺还原酶催化2-吡啶-1-吡咯啉生成(S)-2-(3-吡啶)-吡咯烷还原生成(S)-去甲烟碱，底物浓度达到0.4M/L(58.4g/L)，提高底物浓度，转化率明显下降[US 10913962.B2]。
由于胺类底物独特的性质，导致在还原过程中底物浓度往往不能过高，否则反应转化率会明显降低，昂贵辅酶的NAD(P)H的用量也较高，因此寻找更高活性，高底物耐受性的亚胺还原酶通成为了高效还原亚胺底物2-吡啶-1-吡咯啉生成(S)-去甲烟碱的关键因素。
发明内容
为了在温和的反应条件下高效合成(S)-去甲烟碱，本发明对来源于Myxococcusfulvus的亚胺还原酶MfIR(WP_074958336.1)进行定向进化，获得酶活力及底物耐受提高的亚胺还原酶突变体，对亚胺进行还原从而制备高手性纯度的(S)-2-(3-吡啶)-吡咯烷。
本发明第一方面提供一种合成高手性纯度的(S)-去甲烟碱的方法，利用工程化改造后的亚胺还原酶作为催化剂，改造后的亚胺还原酶与SEQ ID NO.1具有至少90％同一性，且所述的突变体转化产物(S)-去甲烟碱的ee值>99％。
另一方面提供的亚胺还原酶突变蛋白，所述的突变蛋白为非天然蛋白，且所述突变蛋白是在对应于SEQ ID NO.1:1-291中的位点65，123和212在内的一个或多个位点突变的亚胺还原酶突变体：
在另一优选例中，所述的第65位天冬酰胺(N)突变为缬氨酸(V)、脯氨酸(P)，优选缬氨酸(V)。
在另一优选例中，所述的第123位苏氨酸突变为脯氨酸(P)、酪氨酸(Y)、苯丙氨酸(F)、缬氨酸(V)，优选脯氨酸(P)。
在另一优选例中，第212位甲硫氨酸(M)突变为缬氨酸(V)、亮氨酸(L)和异亮氨酸(I)，优选异亮氨酸(I)。
更具体的是下述组合突变：第65位突变为缬氨酸(V)且第212位突变为亮氨酸(L)或异亮氨酸(I)；第123位突变为脯氨酸(P)、酪氨酸(Y)且第212位突变为亮氨酸(L)或异亮氨酸(I)。
第三方面，第二方面所述的亚胺还原酶突变体催化如下反应：具体地，所述催化反应以含亚胺还原酶突变体编码基因工程菌经发酵后的培养物、菌体细胞或破碎液作为催化剂，以麦斯明为底物，以NADP(H)为辅酶，以pH为6.0-10.0的缓冲液为反应介质，在25℃-50℃、搅拌条件下进行还原反应；
所述反应具有选自下组的一个或多个特点：
(i)反应体系含有菌体量为10-50g/L，更佳地，10-30g/L；
(ii)反应体系的pH为6.0-10.0，较佳地，6.0-8.0，更佳地，7.5；
(iii)反应体系温度为20℃-50℃，较佳地，20℃-30℃，更佳地，25℃。
(iv)催化底物浓度为5-150g/L，更佳地，底物浓度为80-130g/L；
(v)催化获得的(S)-去甲烟碱的ee值≥99.0％。
附图说明
图1显示了MfIR与突变体蛋白经诱导表达后结果。其中，M表示Marker，1为野生型MsIR1可溶蛋白表达，2-6为代表性突变体可溶蛋白表达。
图2产物去甲烟碱消旋体的HPLC谱图。
图3(S)-去甲烟碱的HPLC谱图。
具体实施方式
下面的实施例进一步说明本发明的内容，但不应理解为对本发明的限制。
如本文所用，术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，例如N65V表示第65位的氨基酸N突变为V，以此类推。
在本发明的一优选实施方式中，本发明的亚胺还原酶突变体的制备方法如下：大肠杆菌作为表达宿主。具体地，该制备方法包括以下步骤：(1)亚胺还原酶MsIR1相应突变位点的基因构建到pET 28a表达载体上，获得带有目的酶基因的重组质粒。(2)将重组质粒转入宿主菌细胞，优选大肠杆菌BL21(DE3)，获得相应的工程菌株。(3)将工程菌株接种至种子培养基培养，按一定比例接种到发酵培养基，培养一定时间后，加入诱导剂IPTG或乳糖或二者混合物诱导培养一定时间。(4)离心收集菌体，进行高压破碎。
实施例1亚胺还原酶MfIR突变体库的构建
以与MsIR1序列同源性为64％的蛋白(PDB ID：6to4)为模板，通过同源建模获得MsIR1的模拟蛋白结构，选择其底物结合口袋内非保守残基分别进行饱和突变，采用简并密码子NNK设计突变引物，以pET28a-MsIR1作为模板。将所得到的单克隆菌落挑取到96孔深孔板中进行培养，对表达的蛋白进行高通量活力筛选，筛选方法为检测340nm处NADPH的下降。其中反应的具体方法是：底物浓度100mM，NADPH 0.25mg/mL，粗酶液20μL，磷酸钠缓冲液补齐至200μL，酶标仪检测340nm处NADPH的下降，若酶活相对较高，则NADPH的消耗则快，下降曲线的斜率较大。
建库突变的位点分别为13，65，95，120，121，123，124，127，171，174，175，178，179，212，216，236，241和242，获得酶活力提高的有益突变位点有65，123和212，分别为突变体1-8。
实施例2亚胺还原酶MfIR组合突变体库构建
根据饱和突变结果，选取活力提高明显的位点123和212，构建两点的组合突变体库，获得活力进一步提升的突变体9、10、11和12，蛋白序列如SEQ ID NO.:2-5所示。对底物麦斯明建立转化反应考察突变体的转化率及立体选择性，底物浓度为100mM，菌液不浓缩，反应2h检测，酶活测定及转化反应结果见表1。
表1 MfIR及其突变体的酶活力及对底物的转化率及立体选择性
实施例3：亚胺还原酶突变体的诱导表达
配置种子液50mL，培养基为LB液体培养基(蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L)，用接种环挑取基因工程菌单菌落接入培养基中，37℃，200rpm培养过夜。将过夜培养的种子液以1％的接种量转接到发酵培养基，37℃，200rpm培养至OD600为0.6-0.8左右，加入0.1mM的IPTG，诱导10h以上，以8000rpm离心培养液收集菌体，进行高压破碎，得到亚胺还原酶的粗酶液。SDS-PAGE电泳图谱显示MsIR1与突变体的诱导表达情况，如图1所示，其中，M表示Marker，1为MfIR野生型蛋白表达上清，2-6依次为突变体2-6的蛋白表达上清，结果表明，本实施例的方法获得的突变体蛋白与野生型的可溶性蛋白表达量没有变化。
实施例4:利用亚胺还原酶野生型全细胞催化合成(S)-去甲烟碱
磷酸缓冲液(pH 7.5)10ml，菌体30-40mg/ml，2eq葡萄糖,NADP+0-0.5mg/ml,GDH粉末10mg，底物浓度10-90mg/ml，28℃反应，TLC点板判断反应进程。24小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
实施例5利用亚胺还原酶野生型的粗酶液催化合成(S)-去甲烟碱
磷酸缓冲液(pH 7.5)10ml，亚胺还原酶菌体破菌粗酶液30-40mg/ml，2eq葡萄糖,NADP+0-0.5mg/ml,GDH粉末10mg，底物浓度10-90mg/ml，30℃反应，TLC点板判断反应进程。24小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
| 底物浓度 | 辅酶NADP+ | 转化率 | ee | |
| 1 | 10 | 0 | 21.6 | 99.7 |
| 2 | 10 | 0.2 | 99.2 | 99.8 |
| 3 | 20 | 0.2 | 97.8 | 99.7 |
| 4 | 30 | 0.3 | 94.5 | 99.8 |
| 5 | 30 | 0.4 | 99.3 | 99.8 |
| 6 | 40 | 0.4 | 99.2 | 99.7 |
| 7 | 50 | 0.4 | 83.4 | 99.7 |
| 8 | 50 | 0.5 | 89.4 | 99.7 |
| 9 | 60 | 0.5 | 88.5 | 99.7 |
| 10 | 70 | 0.5 | 76.3 | 99.7 |
| 11 | 80 | 0.5 | 64.9 | 99.7 |
实施例6利用亚胺还原酶的全细胞催化合成(S)-去甲烟碱
磷酸缓冲液10ml，菌体25mg/ml，1.2eq葡萄糖,NADP+0.1mg/ml,GDH粉末10mg，底物浓度50mg/ml，28℃反应，TLC点板判断反应进程。12小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
实施例7:利用亚胺还原酶突变体11的全细胞催化合成(S)-去甲烟碱
10ml磷酸缓冲液(pH7.5)，20mg/ml菌体，2eq葡萄糖,0.1mg/ml NADP+,10mg GDH粉末，底物浓度50mg/ml，28℃反应，TLC点板判断反应进程。12小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
| 底物浓度 | 辅酶NADP+ | 转化率 | ee | |
| 1 | 10 | 0 | 21.6 | 99.7 |
| 2 | 10 | 0.2 | 99.2 | 99.8 |
| 3 | 40 | 0.2 | 99.4 | 99.7 |
| 4 | 80 | 0.3 | 99.5 | 99.8 |
| 5 | 120 | 0.3 | 99.3 | 99.8 |
| 6 | 160 | 0.3 | 99.2 | 99.7 |
| 7 | 120 | 0.4 | 99.3 | 99.7 |
| 8 | 130 | 0.4 | 99.2 | 99.7 |
| 9 | 140 | 0.4 | 99.5 | 99.7 |
| 10 | 150 | 0.4 | 98.5 | 99.7 |
| 11 | 160 | 0.4 | 90.6 | 99.7 |
实施例8：利用亚胺还原酶突变体11的全细胞催化合成(S)-去甲烟碱
10ml磷酸缓冲液(pH 7.5)，30-40mg/ml菌体，2eq葡萄糖,0-0.5mg/ml NADP+,10mgGDH粉末，底物浓度10-90mg/ml，28℃反应，TLC点板判断反应进程。24小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
实施:9：利用亚胺还原酶突变体11的粗酶液催化合成(S)-去甲烟碱
10ml磷酸缓冲液(pH 7.5)，30-40mg/ml亚胺还原酶菌体破菌粗酶液，2eq葡萄糖,0-0.5mg/ml NADP+,10mg GDH粉末，底物浓度10-150mg/ml，30℃反应，TLC点板判断反应进程。24小时后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品，HPLC检测。
实施例10：利用亚胺还原酶突变体11的全细胞催化合成(S)-去甲烟碱
1L磷酸缓冲液(pH 7.5)，亚胺还原酶菌体30mg/ml，1.2eq葡萄糖,NADP+0.4mg/ml,GDH100mg粉末，分批补加底物，底物终浓度100mg/ml，28℃反应，TLC点板判断反应进程。反应完全后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品。收率96％，纯度98％，e.e.值99.8％。
实施例11：利用亚胺还原酶突变体11的全细胞催化合成(S)-去甲烟碱
1L磷酸缓冲液(pH 7.5)，亚胺还原酶菌体30mg/ml，1.2eq葡萄糖,NADP+0.5mg/ml,GDH100mg粉末，分批补加底物，底物终浓度120mg/ml，30℃反应，TLC点板判断反应进程。反应完全后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品。收率92％，纯度98％，e.e.值99.6％。
实施例12利用亚胺还原酶突变体11的粗酶液催化合成(S)-去甲烟碱
1L磷酸缓冲液(pH 7.5)，亚胺还原酶菌体30mg/ml破菌粗酶液，1.2eq葡萄糖,NADP+0.3mg/ml,GDH粉末100mg，分批补加底物，底物终浓度150mg/ml，30℃反应，TLC点板判断反应进程。反应完全后，加氢氧化钠饱和溶液调节pH值至10以上，离心除去变性的蛋白，上清液用二氯甲烷萃取，干燥，旋干收集产品。收率94％，纯度98.5％，e.e.值99.7％。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 中国科学院天津工业生物技术研究所
<130> 亚胺还原酶突变体及其应用
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<213> Myxococcus fulvus
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Claims (10)
1.一种亚胺还原酶突变蛋白，其特征在于，所述突变蛋白在对应于SEQ ID NO.1所示氨基酸序列第65位点的谷氨酰胺突变为缬氨酸；第123位点的苏氨酸突变为脯氨酸、酪氨酸、苯丙氨酸、缬氨酸；第212位点的甲硫氨酸突变为缬氨酸、亮氨酸或异亮氨酸。
2.一种亚胺还原酶突变蛋白，其特征在于，所述突变蛋白在对应于SEQ ID NO.1所示氨基酸序列存在如下位点的突变：第123位突变为脯氨酸或酪氨酸且第212位突变为亮氨酸或异亮氨酸。
3.如权利要求1或2所述的亚胺还原酶突变蛋白的编码基因。
4.含有如权利要求1或2所述的亚胺还原酶突变蛋白的编码基因的表达载体。
5.含有如权利要求1或2所述的亚胺还原酶突变蛋白的编码基因的重组细胞。
6.如权利要求1或2所述的亚胺还原酶突变蛋白在制备手性(S)-去甲烟碱中的应用
7.如权利要求6所述的应用，其特征在于，所述应用是以麦斯明(CAS:532-12-7)为底物催化反应合成(S)-去甲烟碱。
8.如权利要求7所述的应用，其特征在于，所述催化反应是以表达所述亚胺还原酶蛋白的编码基因工程菌经发酵培养获得的湿菌体为催化剂，以麦斯明(CAS:532-12-7)为底物，以pH为6.0-10.0的缓冲液为反应介质，在反应温度20℃-50℃的反应温度下进行还原反应。
9.如权利要求8所述的应用，其特征在于，所述催化反应中，反应体系中的催化底物浓度为5-150g/L，含有菌体量为10-50g/L，反应体系的pH为6.0-10.0，反应温度为25℃-35℃。
10.如权利要求8所述的应用，其特征在于，在反应体系中加入NADP+，葡萄糖，葡萄糖脱氢酶，且在150rpm-250rpm的条件下反应，反应时间为5-25小时。
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