CN113774036B - 一种亚胺还原酶突变体及其应用 - Google Patents
一种亚胺还原酶突变体及其应用 Download PDFInfo
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Abstract
本发明涉及一种亚胺还原酶突变体在合成烟碱关键手性中间体(S)‑3‑(吡咯烷‑2‑基)吡啶中的应用，属于生物催化合成技术领域。本发明提供了一种改进的亚胺还原酶，即亚胺还原酶突变体，其氨基酸序列是SEQ ID NO:1所示氨基酸序列（对应编码基因的核苷酸序列为SEQ ID NO:5）的突变体，可选的突变点位至少包括下列点位之一：第246位A或第285位D突变为V。该亚胺还原酶突变体的酶活性高于现有技术，提高了催化还原反应的产率，缩短了反应时间。
Description
技术领域
本发明涉及一种亚胺还原酶突变体在合成烟碱关键手性中间体(S)-3-(吡咯烷-2-基)吡啶中的应用，属于生物催化合成技术领域。
背景技术
烟碱（Nicotine）又称尼古丁，化学名为：1-甲基-2-(3-吡啶基)吡咯烷，是一种天然生成的液态生物碱，在烟草工业、精细化工、制药、有机合成、农业等有着重要的用途。尤其是高纯度烟碱，已成为国际市场上紧俏产品之一。(S)-3-(吡咯烷-2-基)吡啶作为烟碱合成中的关键手性中间体，目前已报道的制备工艺中最具优势的是利用亚胺还原酶催化3-（1-吡咯啉-2-基）吡啶进行制备，可以达到较高的光学纯度。
专利WO2020098978 A1公开了使用亚胺还原酶还原3-（1-吡咯啉-2-基）吡啶，具体公开了9种可用于还原3-（1-吡咯啉-2-基）吡啶的亚胺还原酶，以葡萄糖脱氢酶/葡萄糖为辅酶再生系统，进行(S)-原烟碱的催化反应，这些亚胺还原酶包括：IRED-A、IRED-B、IRED-C、IRED-D、 IRED-E、 IRED-F、IRED-P、IRED-X、IRED-AB。IRED-C在3-（1-吡咯啉-2-基）吡啶浓度为400 mmol/L（即58 g/L）时，24h转化率达99.6%，e.e值为99.8%；IRED-C在3-（1-吡咯啉-2-基）吡啶浓度为1 mol/L（即146 g/L）时，24h转化率仅52.4%，e.e值为99.6%。其对3-（1-吡咯啉-2-基）吡啶的催化能力较差，不利于放大生产，生产成本也偏高。寻找不同生物来源的亚胺还原酶，并借助蛋白质工程方法对酶分子进行改造获得更加高效的亚胺还原酶，是实现烟碱关键手性中间体(S)-3-(吡咯烷-2-基)吡啶酶法工业化生产的努力方向。
发明内容
发明目的：针对现有生物法制备(S)-3-(吡咯烷-2-基)吡啶的不足，提供一种适合工业化生产的制备工艺，为社会提供质优价廉的原料药。
技术方案：本发明基于Nocardiopsis alba菌株的天然亚胺还原酶而改进的亚胺还原酶及其编码基因，利用亚胺还原酶突变体催化前体3-（1-吡咯啉-2-基）吡啶合成(S)-3-(吡咯烷-2-基)吡啶，反应式如下：
式中：NAD是生物学专有名词，中文名：烟酰胺腺嘌呤二核苷酸，简称为辅酶；（P）表示括号内P可有可无，当有时，为NADP。
NADP是烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotidephosphate)的缩写，曾称为三磷酸吡啶核苷酸（TPN）或辅脱氢酶Ⅱ或氧化型辅酶Ⅱ。
本发明提供了一种改进的亚胺还原酶，即亚胺还原酶突变体，该亚胺还原酶突变体的酶活性和立体选择性均高于野生型亚胺还原酶和专利WO2020098978 A1中的IRED-C。
本发明的技术方案是，一种亚胺还原酶突变体，其氨基酸序列是SEQ ID NO: 1所示氨基酸序列（对应编码基因的核苷酸序列为SEQ ID NO: 5）的突变体，可选的突变点位至少包括下列点位之一：第246位A或第285位D突变为V。
本发明所述亚胺还原酶突变体的氨基酸序列为：SEQ ID NO: 2、SEQ ID NO: 3或SEQ ID NO: 4所示，对应编码基因的核苷酸序列分别为SEQ ID NO: 6、SEQ ID NO: 7或SEQID NO: 8所示。
根据本发明的另一方面，提供了一种重组质粒，重组质粒上含有上述任一种基因的核苷酸序列，进一步地，质粒为pET-28a(+)、pET-28b(+)、pET-28c(+)、pET-5b(+)、pET-15b、pET-24a(+)、pET-24c(+)、pET-24d(+)、pET-25b(+)、pET-27b(+)、pET-28c(+)、pET-29a(+)、pET-29b(+)、pET-29c(+)、pET-30b(+)、pET-30c(+)、pET-30 Xa/LIC、pET-30 EK/LIC、pET-31b(+)、pET-32b(+)、pET-32c(+)、pET-32 EK/LIC、pET-32 Xa/LIC、pET-33b(+)、pET-37b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42b(+)、pET-42c(+)、pET-43.1a(+)、pET-43.1b(+)、pET-43.1c(+)、pET-43.1 EK/LIC、pET-44a(+)、pET-44b(+)、pET-44c(+)、pET-44 EK/LIC、pET-45b(+)、pET-46 EK/LIC、pET-47b(+)、pET-48b(+)、pET-49b(+)、pET-51b(+)、pET-52b(+)、pQE30、pQE31、pQE32、pQE40、pBV220、pBV221、pCold-GST、pCold IV、pCold-GST或pTrcHis C等。
根据本发明的另一方面，提供了一种宿主细胞，宿主细胞内含有上述任一种重组质粒，且所述宿主细胞包括原核细胞或真核细胞，原核细胞优选为大肠杆菌BL21(DE3)细胞。
根据本发明的另一方面，还提供了亚胺还原酶突变体在制备(S)-3-(吡咯烷-2-基)吡啶中的应用，包括：在亚胺还原酶突变体存在下，以3-（1-吡咯啉-2-基）吡啶为底物，经不对称催化加氢获得(S)-3-(吡咯烷-2-基)吡啶。
具体的，上述亚胺还原酶突变体在制备(S)-3-(吡咯烷-2-基)吡啶中的应用，催化加氢反应用磷酸盐缓冲溶液控制pH在6.8-7.8范围，优选7.0-7.2，用氢氧化钠调整反应过程中的pH。
反应过程中，pH低于6.8，或高于pH8.0，酶催化反应速度和收率明显降低。
具体的，上述亚胺还原酶突变体在制备(S)-3-(吡咯烷-2-基)吡啶中的应用，催化加氢反应温度控制在20-35℃，优选25-30℃。反应温度低于20℃或高于40℃，酶催化反应速度会有所降低。
附图说明：
图1. 亚胺还原酶及突变体催化3-（1-吡咯啉-2-基）吡啶的不对称还原反应。
图2. 本发明优选实施例6中亚胺还原酶突变体的蛋白电泳检测结果图：其中，1表示野生型亚胺还原酶母本；2表示A246V突变体；3表示D285V突变体；4表示A246V-D285V突变体。
有益效果：本发明技术方案通过在SEQ ID NO: 1所示的野生型亚胺还原酶（对应编码基因的核苷酸序列如SEQ ID NO: 5所示）的基础上，采用随机突变的分子生物学方法对亚胺还原酶的基因进行突变，从而改变酶的氨基酸序列，实现酶结构和功能的改变，再通过定向筛选的方法，得到具有上述位点中的至少之一发生突变的亚胺还原酶。使用该亚胺还原酶突变体催化3-（1-吡咯啉-2-基）吡啶合成(S)-3-(吡咯烷-2-基)吡啶，相比于野生型亚胺还原酶母本，酶活性提高为约12倍（实施例5），具有非常高的立体选择性和转化率，表现在100 g/L 3-（1-吡咯啉-2-基）吡啶，0.3 wt亚胺还原酶粗酶液（按照细胞湿重计算），反应4 h，底物转化率可达到99.5%以上，产物e.e.值达到99.3%，相比于现有公开的最优酶催化工艺（专利WO2020098978 A13-（1-吡咯啉-2-基）吡啶浓度为58.4g/L时，24h转化率达99.6%，e.e值为99.8%），本发明提供了一种底物浓度更高（1.7倍）、底物转化率（反应4h相对于反应24h）更高的技术方案，可进一步降低(S)-3-(吡咯烷-2-基)吡啶和烟碱的工业生产成本，具有良好的工业应用价值。
具体实施方式：下面结合具体实施实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1：获得Nocardiopsis alba菌株的野生型亚胺还原酶母本重组质粒
通过NCBI GenBank核酸数据库获得Nocardiopsis alba菌株的亚胺还原酶母本编码基因（GenBank: AFR08535.1），经密码子优化并委托服务商人工合成全长基因至pET21a(+)表达质粒，后转化大肠杆菌BL21(DE3)感受态细胞，涂布于含有100 mg/L 氨苄青霉素的LB琼脂平皿中，37°C培养过夜。挑选数个单菌落至LB培养基（含100 mg/L 氨苄青霉素），37°C培养过夜后使用质粒小提试剂盒提取重组质粒，进行PCR和测序验证，获得亚胺还原酶母本的重组质粒。
实施例2：对野生型亚胺还原酶母本基因进行随机突变
根据实施例1所述内容，以含Nocardiopsis alba菌株的亚胺还原酶母本编码基因的重组质粒为模板，并根据Nocardiopsis alba菌株的亚胺还原酶母本编码基因用Primer5.0设计并合成两端引物（表1），使用易错PCR技术（物料及浓度见表2，反应条件见表3）获得含有大量碱基突变的线性基因片段，将这些PCR产物和pET21a(+)表达质粒分别进行酶切、切胶回收、连接和转化大肠杆菌BL21(DE3)感受态细胞，涂布于含有100 mg/L 氨苄青霉素的LB琼脂平皿中，37°C培养过夜。详见表1-表3。
表1. 随机突变引物序列
表2. 50 μL易错PCR物料体系
表3. 易错PCR反应条件
实施例3：亚胺还原酶突变体的克隆与表达
为了便于亚胺还原酶突变体的克隆、表达及鉴定，在其基因的5’和3’末端设计了兼容的限制内切酶位点，可以采用Nde I和Xho I限制性内切酶分别将目的基因和pET21a(+)（其它可以在大肠杆菌中表达蛋白质的表达质粒也可使用）同时进行酶切和DNA切胶回收，回收后的目的基因和质粒的较大片段用T4 DNA连接酶进行连接反应，将连接产物转化到大肠杆菌BL21(DE3)感受态细胞中，然后将转化后的感受态细胞涂布于含有100 mg/L 氨苄青霉素的LB琼脂平皿中，37°C培养过夜。
挑取上述培养皿上长出的单菌落接种于含有100 mg/L 氨苄青霉素的LB 液体培养基中，37°C振荡培养过夜，收集菌体进行质粒提取、PCR 鉴定和双酶切鉴定后，将正确的重组质粒命名为pET21a(+)-A-N，并将含有正确的重组质粒的大肠杆菌进行后续的诱导表达。将上述菌液转接于500 mL含100 mg/L 氨苄青霉素的LB 液体培养基中，37°C振荡培养至OD600＝0.6～0.8 时，加入IPTG 至终浓度分别为0.05～0.5 mM，在22～25°C下进行诱导表达12～16 h后，取出菌液，6000 ×g 离心20 min收集菌体，于-20°C冻存备用。
实施例4：亚胺还原酶突变体的初筛
根据实施例2和实施例3所述内容，挑取上述LB琼脂培养基上的单克隆菌落接种于96深孔板中，每孔预先加入1 mL含有100 mg/L 氨苄青霉素的LB培养基，于37°C，220 rpm振荡培养3 h后，加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG，终浓度为0.1 mM），25°C ，220 rpm诱导培养16 h，6000 ×g离心20 min收集菌细胞，倒掉离心上清液后使用相同体积的100 mM磷酸盐缓冲液（pH 7.0）重悬菌体。利用宁波新芝生物股份有限公司生产的高通量超声波细胞粉碎机超声破碎菌体（40%功率，工作3 s，间隙3 s，总时长10 min），于4°C，6000 ×g离心20 min获得上清液，即亚胺还原酶突变体粗酶液，用酶标仪进行活性初筛。向96孔板中加入20 μL 2 mmol/L还原型烟酰胺腺嘌呤二核苷酸（NADH）溶液和140μL的磷酸盐缓冲液（100 mM，pH=7.0），20μL酶液， 20 μL 20mmol/L 3-（1-吡咯啉-2-基）吡啶溶液，检测5min内，每隔10s A340的变化。
酶活计算公式：酶活（U/mL）=（△A×V1×103）/（6220×t×V2）
△A：t内吸光度的变化值；
V1：反应体系的总体积，mL；
V2：加入的酶液体积，mL；
6220：摩尔消光系数，L/mol/cm；
t：检测时间。
实施例5：亚胺还原酶突变体的复筛
（1）亚胺还原酶突变体酶液的制备
将实施例4中酶活高于母本的突变体菌株分别以0.1%接种量接种于500 mL含有100 mg/L 氨苄青霉素的LB培养基，于37℃，220 rpm振荡培养5~6 h，加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG，终浓度为0.1 mM），25℃，220 rpm诱导培养16 h，6000 ×g离心收集菌体。使用100 mM磷酸盐缓冲液（pH 7.0）重悬菌体后用超声波破碎仪破碎细胞，4℃，6000 ×g离心20 min获得上清液，即亚胺还原酶突变体粗酶液。
（2）亚胺还原酶催化3-（1-吡咯啉-2-基）吡啶制备(S)-3-(吡咯烷-2-基)吡啶的反应
向10 mL反应瓶中加入0.1 g主原料3-（1-吡咯啉-2-基）吡啶，加入2 mL磷酸盐缓冲液（100 mM ，pH =7.0），加入2 mg NADP+，0.5 mL副酶（GDH，葡萄糖脱氢酶）粗酶液和适量上述突变体粗酶液，控制反应液pH为7.0，并于30℃反应6 h后， HPLC分析其转化率及e.e值。
（3）亚胺还原酶催化转化率的测定
在（2）所述的反应体系经甲醇（反应体系:甲醇=1:9）处理，膜过滤后HPLC直接进样分析。HPLC条件为：
仪器：Thermo U3000 system HPLC
色谱柱：Agilent poroshell 120 C18， 4.6mm×150mm，4μm
流动相：以0.01mol/L磷酸氢二钾（用磷酸调pH7.8）为流动相A，乙腈为流动相B，按表4进行线性梯度洗脱；
表4.
检测波长：260 nm；
流速：1.0 mL/min；
柱温：40℃；
进样体积：10μL。
转化率计算公式：
式中，A（P）为(S)-3-(吡咯烷-2-基)吡啶峰面积；
A（S）为起始物料3-（1-吡咯啉-2-基）吡啶峰面积。
（4）亚胺还原酶催化产物光学纯度鉴定
在（2）所述的反应体系经流动相（反应体系:流动相=1:50）处理，膜过滤后HPLC直接进样分析。HPLC条件为：
仪器：Thermo U3000 system HPLC
色谱柱：Chiralpak AD-H，4.6mm×250mm，5μm
流动相：正己烷 : 乙醇 : 二乙胺=90:10:0.1
检测波长：260 nm；
流速：1.0 mL/min；
柱温：25℃；
进样体积：20μL。
S型产物光学纯度计算公式：
式中，A（S）为目标产物(S)-3-(吡咯烷-2-基)吡啶峰面积；
A（R）为对映异构体(R)-3-(吡咯烷-2-基)吡啶峰面积。
选取催化活性优于母本的突变体进行测序，分析突变位点，复测催化活性确定突变体A246V （SEQ ID NO: 2，对应编码基因的核苷酸序列为SEQ ID NO: 6）、D285V（SEQ IDNO: 3，对应编码基因的核苷酸序列为SEQ ID NO: 7）、A246V-D285V（SEQ ID NO: 4，对应编码基因的核苷酸序列为SEQ ID NO: 8）的催化活性及立体选择性均比本方案母本显著提高，复筛反应结果如表5所示。
表5. 亚胺还原酶母本与突变体酶法制备(S)-3-(吡咯烷-2-基)吡啶活性比较
注：表5中的*指转化1 g底物所需各亚胺还原酶重组细胞的湿重。1wt 指转化1 g主原料需要1 g亚胺还原酶突变体重组湿细胞。
表4结果说明：A246V-D285V突变体为最优亚胺还原酶突变体，其对3-（1-吡咯啉-2-基）吡啶的催化效率为野生型亚胺还原酶母本的约12倍，且产物有更高的e.e.值。
实施例6：亚胺还原酶突变体酶液制备
将实施例5中突变体菌株分别以0.01%接种量接种于（500 mL/瓶*10瓶）含有100mg/L 氨苄青霉素的LB培养基，于37°C，220 rpm振荡培养5~6 h，加入一定量的诱导剂异丙基-β-D-硫代半乳糖苷（IPTG，终浓度为0.1 mM），25°C，220 rpm诱导培养16 h，6000 ×g离心收集菌体。所得30~40 g菌细胞使用150 mL 100 mM磷酸盐缓冲液（pH 7.0）重悬菌细胞后用高压均质机破碎细胞，4°C，6000 ×g离心20 min获得上清液即亚胺还原酶突变体酶液。
实施例7： SEQ ID NO:4所示的亚胺还原酶突变体在(S)-3-(吡咯烷-2-基)吡啶制备中的应用
在1000 mL反应瓶中加入100 g主原料3-（1-吡咯啉-2-基）吡啶，480 mL磷酸盐缓冲液（100 mM，pH 7.0），150 mL 亚胺还原酶粗酶液，150 mL葡萄糖脱氢酶粗酶液，204 g一水合葡萄糖，157 mg氧化型辅酶II二钠（NADP-Na2），25±3℃反应4 h，反应过程用2 mol/LNaOH溶液调节反应pH为7.2。4 h反应转化率为99.9%。反应体系使用稀盐酸调pH 3.0±0.5后，加入活性炭30±5 g于70±5℃热处理30~60 min，加入50 g硅藻土过滤体系得(S)-3-(吡咯烷-2-基)水溶液，外标法测得(S)-3-(吡咯烷-2-基)为91.5 g，收率90%，产物e.e.值为99.3%。
结果表明，SEQ ID NO:4所示的亚胺还原酶突变体在酶催化3-（1-吡咯啉-2-基）吡啶中，即100 g/L底物和0.3wt粗酶液（按照细胞湿重计算）的反应体系中，反应4 h，转化率可达到99.9%以上，产物e.e.值达到99.3%，筛选出的亚胺还原酶突变体在酶法制备(S)-3-(吡咯烷-2-基)吡啶均表现出了极高的立体选择性和高效性。
对照例1： SEQ ID NO:4所示的亚胺还原酶突变体在(S)-3-(吡咯烷-2-基)吡啶制备中的应用
在1000 mL反应瓶中加入100 g主原料3-（1-吡咯啉-2-基）吡啶，480 mL磷酸盐缓冲液（100 mM，pH 7.0），150 mL 亚胺还原酶粗酶液，150 mL葡萄糖脱氢酶粗酶液，204 g一水合葡萄糖，157 mg氧化型辅酶II二钠（NADP-Na2），25±3℃反应4 h，反应过程用2 mol/LNaOH溶液调节反应pH为8.5。4 h反应转化率为23.4%，24 h反应转化率为35.5%。反应体系使用稀盐酸调pH 3.0±0.5后，加入活性炭30±5 g于70±5℃热处理30~60 min，加入50 g硅藻土过滤体系得(S)-3-(吡咯烷-2-基)水溶液，外标法测得(S)-3-(吡咯烷-2-基)为36.2g，收率35.7%，产物e.e.值为99.2。
结果说明，催化反应pH在8.5情况下，虽然产物e.e.值未受影响，但是一会影响酶催化反应速度，二会影响产品收率。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
本发明所述氨基酸及核苷酸序列表
<110> 迪嘉药业集团有限公司
<120> 一种亚胺还原酶突变体及其应用
<130> 20210820
<141> 2021-08-21
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Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
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Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
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Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
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Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
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Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
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Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
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Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
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Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Ala Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
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Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Asp Arg Ile Tyr
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Glu Glu Leu Arg Ala Gly
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Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
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Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
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Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Val Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Asp Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 3
<211> 294
<212> PRT
<213> D285V Mutant
<400> 3
Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
1 5 10 15
Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Ala Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Val Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 4
<211> 294
<212> PRT
<213> A246V-D285V Mutant
<400> 4
Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
1 5 10 15
Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Val Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Val Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 5
<211> 885
<212> DNA
<213> Nocardiopsis alba Wild type
<400> 5
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgcgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgatcgtat ttatgaggaa ctgcgtgcgg gttaa 885
<210> 6
<211> 885
<212> DNA
<213> A246V Mutant
<400> 6
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgtgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgatcgtat ttatgaggaa ctgcgtgcgg gttaa 885
<210> 7
<211> 885
<212> DNA
<213> D285V Mutant
<400> 7
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgcgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgttcgtat ttatgaggaa ctgcgtgcgg gttaa 885
<210> 8
<211> 885
<212> DNA
<213> A246V-D285V Mutant
<400> 8
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgtgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgttcgtat ttatgaggaa ctgcgtgcgg gttaa 885
序列表
<110> 迪嘉药业集团有限公司
<120> 一种亚胺还原酶突变体及其应用
<130> 20210820
<141> 2021-08-21
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 294
<212> PRT
<213> Nocardiopsis alba Wild type
<400> 1
Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
1 5 10 15
Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Ala Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Asp Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 2
<211> 294
<212> PRT
<213> A246V Mutant
<400> 2
Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
1 5 10 15
Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Val Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Asp Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 3
<211> 294
<212> PRT
<213> D285V Mutant
<400> 3
Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
1 5 10 15
Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Ala Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Val Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 4
<211> 294
<212> PRT
<213> A246V-D285V Mutant
<400> 4
Met Met Lys Asn Asp Gly Val Thr Lys Gly Ser Val Ala Leu Leu Gly
1 5 10 15
Leu Gly Glu Met Gly Arg Val Leu Ala Glu Arg Leu Leu Asp Ala Gly
20 25 30
Tyr Pro Val Thr Val Trp Asn Arg Thr Pro Gly Arg Asp Thr Ala Leu
35 40 45
Val Glu Arg Gly Ala Arg Arg Ala Glu Thr Val Arg Glu Ala Val Thr
50 55 60
Ala Ala Thr Thr Val Val Thr Cys Leu Phe Asp His Ala Ser Val Arg
65 70 75 80
Glu Thr Leu Glu Pro Val Gly Ala Asp Leu Ala Gly Arg Thr Leu Val
85 90 95
Asp Leu Thr Thr Thr Thr Pro Asn Glu Ala Arg Trp Leu Gly Gly Trp
100 105 110
Ala Glu Glu Arg Gly Ile Glu His Leu Asp Gly Ala Ile Met Ala Thr
115 120 125
Pro Ser Met Ile Gly Ala Pro Glu Ala Ser Leu Leu Tyr Ser Gly Ser
130 135 140
Ala Glu Ala Phe Gly Arg His Arg Thr Leu Phe Glu Val Trp Gly Ser
145 150 155 160
Ala Thr Tyr Asp Gly Ala Asp His Gly Ala Ala Ser Leu Phe Asp Leu
165 170 175
Ala Leu Leu Ser Gly Met Tyr Thr Met Phe Thr Gly Phe Ala His Gly
180 185 190
Ala Ala Met Val Thr Ser Ala Gly Val Thr Ala Glu Glu Phe Ala His
195 200 205
Arg Ser Ala Arg Leu Leu Ser Ala Met Thr Gly Val Phe Pro Met Thr
210 215 220
Ala Lys Val Ile Asp Glu Gly Asp Tyr Thr Gly Pro Gly Gln Ser Leu
225 230 235 240
Glu Trp Thr Ala Thr Val Leu Asp Thr Ile Ala Arg Ala Ser Ala Glu
245 250 255
Gln Gly Val Ser Pro Gly Pro Ile Glu Met Thr Arg Ala Leu Val Leu
260 265 270
Ala Gln Ile Glu Ala Gly Tyr Gly Asn Glu Asn Ser Val Arg Ile Tyr
275 280 285
Glu Glu Leu Arg Ala Gly
290
<210> 5
<211> 885
<212> DNA
<213> Nocardiopsis alba Wild type
<400> 5
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgcgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgatcgtat ttatgaggaa ctgcgtgcgg gttaa 885
<210> 6
<211> 885
<212> DNA
<213> A246V Mutant
<400> 6
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgtgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgatcgtat ttatgaggaa ctgcgtgcgg gttaa 885
<210> 7
<211> 885
<212> DNA
<213> D285V Mutant
<400> 7
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgcgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgttcgtat ttatgaggaa ctgcgtgcgg gttaa 885
<210> 8
<211> 885
<212> DNA
<213> A246V-D285V Mutant
<400> 8
atgatgaaga acgacggtgt gaccaaaggc agcgttgcgc tgctgggtct gggtgaaatg 60
ggtcgtgttc tggcggaacg tctgctggat gcgggttacc cggtgaccgt ttggaaccgt 120
accccgggtc gtgataccgc gctggttgaa cgtggcgcgc gtcgtgcgga gaccgtgcgt 180
gaagcggtta ccgcggcgac caccgtggtt acctgcctgt tcgaccatgc gagcgtgcgt 240
gagaccctgg aaccggttgg tgcggacctg gcgggtcgta ccctggtgga tctgaccacc 300
accaccccga acgaagcgcg ttggctgggt ggctgggcgg aggaacgtgg tatcgagcac 360
ctggatggcg cgattatggc gaccccgagc atgattggtg cgccggaggc gagcctgctg 420
tacagcggta gcgcggaagc gttcggtcgt caccgtaccc tgtttgaggt ttggggtagc 480
gcgacctacg acggtgcgga tcatggtgcg gcgagcctgt ttgatctggc gctgctgagc 540
ggcatgtata ccatgttcac cggttttgcg catggtgcgg cgatggtgac cagcgcgggc 600
gttaccgcgg aggaatttgc gcaccgtagc gcgcgtctgc tgagcgcgat gaccggtgtg 660
tttccgatga ccgcgaaggt tattgacgaa ggcgattata ccggtccggg tcagagcctg 720
gagtggaccg cgaccgtgct ggacaccatc gcgcgtgcga gcgcggagca gggtgtgagc 780
ccgggtccga ttgaaatgac ccgtgcgctg gttctggcgc aaatcgaggc gggttacggc 840
aacgaaaaca gcgttcgtat ttatgaggaa ctgcgtgcgg gttaa 885
Claims (7)
1.一种亚胺还原酶突变体，其特征在于，所述亚胺还原酶突变体的氨基酸序列是在SEQID NO:1所示的氨基酸序列上发生突变的氨基酸序列，可选的突变点位包括下列点位之一：第246位A突变为V或第246位A和第285位D同时突变为V。
2.一种重组质粒，其特征在于，含有权利要求1所述的亚胺还原酶突变体的编码基因。
3.根据权利要求2所述的重组质粒，其特征在于，所述质粒为pET-28a(+)、pET-28b(+)、pET-28c(+)、pET-5b(+)、pET-15b、pET-24a(+)、pET-24c(+)、pET-24d(+)、pET-25b(+)、
pET-27b(+)、pET-28c(+)、pET-29a(+)、pET-29b(+)、pET-29c(+)、pET-30b(+)、pET-30c(+)、pET-30Xa/LIC、pET-30EK/LIC、pET-31b(+)、pET-32b(+)、pET-32c(+)、pET-32EK/LIC、pET-32Xa/LIC、pET-33b(+)、pET-37b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42b(+)、pET-42c(+)、pET-43.1a(+)、pET-43.1b(+)、pET-43.1c(+)、pET-43.1EK/LIC、pET-44a(+)、pET-44b(+)、pET-44c(+)、pET-44EK/LIC、pET-45b(+)、pET-46EK/LIC、pET-47b(+)、pET-48b(+)、pET-49b(+)、pET-51b(+)、pET-52b(+)、pQE30、pQE31、pQE32、pQE40、pBV220、pBV221、pCold-GST、pColdIV、pCold-GST或pTrcHisC。
4.一种宿主细胞，其特征在于，含有权利要求2所述的重组质粒。
5.根据权利要求4所述宿主细胞，其特征在于，所述宿主细胞选自原核细胞或真核细胞，所述原核细胞为大肠杆菌BL21(DE3)细胞。
6.权利要求1所述的亚胺还原酶突变体在将3-（1-吡咯啉-2-基）吡啶转化为(S)-3-(吡咯烷-2-基)吡啶中的应用。
7.根据权利要求6所述的应用，其特征在于，催化加氢反应溶液pH在6.8-7.8范围。
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