CN114717211A - 亚胺还原酶突变体m5及其在合成氮杂环手性胺中的应用 - Google Patents

亚胺还原酶突变体m5及其在合成氮杂环手性胺中的应用 Download PDF

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CN114717211A
CN114717211A CN202210294265.1A CN202210294265A CN114717211A CN 114717211 A CN114717211 A CN 114717211A CN 202210294265 A CN202210294265 A CN 202210294265A CN 114717211 A CN114717211 A CN 114717211A
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廖道红
高书山
雷晓光
张军
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Jiangsu Jicui Molecule Engineering Research Institute Co ltd
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Abstract

本发明属于还原胺化酶技术领域。本发明提供了一种亚胺还原酶突变体M5，其具有序列表中SEQ I D NO.4所述的氨基酸序列。同时还还提供了还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用。

Description

亚胺还原酶突变体M5及其在合成氮杂环手性胺中的应用
技术领域
本发明属于还原胺化酶技术领域。
背景技术
手性胺在药物、农用化学品和材料有着广泛的应用，近40％的药物含有一个或多个手性胺，因此胺的不对称合成在工业生产中极为重要。还原胺化是合成胺的一类重要的反应，在还原剂存在下，利用前手性酮或醛与胺直接合成烷基化手性胺。不对称还原胺化的传统化学方法通常需要昂贵的过渡金属络合物或手性配体，再加上酮/醛还原或过烷基化等副反应，使其在工业应用过程中大大受限。然而酶催化的还原胺化反应，因其低成本、高选择、高催化效率、绿色无污染和可进化性方面的明显优势。
近年来，Nicholas J.Turner课题组报道了亚胺还原酶(Imine reductase,IRED)可以催化前手性酮或醛与胺进行还原胺化反应，直接合成烷基化手性胺，并且具有很高的底物杂泛性。然而，天然的 IREDs因催化效率低和稳定性差等问题阻碍了其在工业中应用。为了使IREDs适用于工业化应用，应用蛋白质工程技术对其进行改造已成为解决上述问题的必要手段，并且近期报道了两个成功的案例，例如抗白血病药物GSK2879552和Abrocitinib JAK1抑制剂的酶法工业级生产。尽管还原胺化酶已经显现出应用于生产手性胺的希望，但仍有几个问题需要解决，以构建一个可工业化的有利突变体。首先，提供的证据表明还原氨化酶活性是混杂的结果，并且不限于任何活性位点残基，缺乏有前景的工程化热点残留物信息。其次，这种转化需要胺和酮都可以进入酶活性位点以形成亚胺中间体，亚胺的结合模式和动力学性质仍然知之甚少，这给合理工程化带来了重大挑战。第三，RedAms对大的空间位阻底物表现出有限的活性。
发明内容
有鉴于此，本发明提供了一种亚胺还原酶突变体M5，其具有序列表中SEQ ID NO.4所述的氨基酸序列。
本发明还提供了还原胺化酶突变体M5在酮与胺合成烷基化手性胺的反应中的应用，所述酮为
Figure RE-GDA0003659649040000021
其中R1为
Figure RE-GDA0003659649040000022
n为1-10的整数，m为1-10的整数；所述胺为R2-NH2，其中，R2为
Figure RE-GDA0003659649040000023
Figure RE-GDA0003659649040000024
X为1-10的整数。
在本发明的具体实施例中，所述反应中加入酮与胺的物质的量比为1:1.1。
在本发明的具体实施例中，所述反应中加入还原胺化酶突变体M5 的冻干裂解物，所述还原胺化酶突变体M5的冻干裂解物与酮的质量比为4-20％。在本发明的具体实施例中，所述反应中加入D-葡萄糖，所述D-葡萄糖与酮的物质的量比为1.5：1。
在本发明的具体实施例中，所述反应中加入NADP+的量为1 mM/mL；GDH酶粉的量为1.5mg/mL；磷酸钠缓的量为100mM/mL；DMSO, 所述DMSO的体积用量是反应液总体积的20％。
在本发明的具体实施例中，所述反应的溶液pH 7.0。
在本发明的具体实施例中，所述反应的温度为30℃。
在本发明的具体实施例中，所述反应的时间为24小时。
在本发明的具体实施例中，所述反应的步骤包括：将体积比为1:4 的DMSO和磷酸钠缓冲液加入生物反应器中；将胺、D-葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸二钠盐的混合液加入反应器中，在30℃条件下反应；所述胺为苄胺，所述苄胺与酮的物质的量比为1.1:1；将亚胺还原酶突变体M5的冻干酶粉和GDH酶粉末添加到反应溶液中反应；所述亚胺还原酶突变体M5的冻干酶粉与酮的质量比为1：4；反应24 小时后得产物。所述酮为N-Boc-3-哌啶酮，所述N-Boc-3-哌啶酮的加入量为125mM/L。
在本发明的具体实施例中，所述在反应24小时后得产物之后还包括步骤：用乙酸淬灭反应并搅拌以得到pH 3.0的溶液；将硅藻土装入生物反应器并搅拌10分钟；过滤混合物并用水冲洗得滤液；滤液依次用二氯甲烷萃取、用饱和碳酸钠将水相碱化至pH 10、用二氯甲烷萃取得有机相萃取液；减压除去合并的有机萃取物的溶剂。
本发明通过合理的酶空腔设计和热稳定性工程对天然酶进行了改造得到了突变体M5，通过一系列手性氮杂环烷基胺的合成证明了其高选择性、高活性、高立体选择性、高转化率以及优秀的底物相容性。
附图说明
图1.M5催化的还原胺化产物手性HPLC分析图谱。
图2.M5催化的还原胺化产物手性HPLC分析图谱。
图3.M5催化的还原胺化产物手性HPLC分析图谱。
图4.M5催化的还原胺化产物手性HPLC分析图谱。
图5.M5催化的还原胺化产物手性HPLC分析图谱。
图6.M5催化的还原胺化产物手性HPLC分析图谱。
图7.M5催化的还原胺化产物手性HPLC分析图谱。
图8.M5催化的还原胺化产物手性HPLC分析图谱。
图9.M5催化的还原胺化产物手性HPLC分析图谱。
图10.M5催化的还原胺化产物手性HPLC分析图谱。
图11.M5催化的还原胺化产物手性HPLC分析图谱。
图12.M5催化的还原胺化产物手性HPLC分析图谱。
图13.M5催化的还原胺化产物手性HPLC分析图谱。
图14.M5催化的还原胺化产物手性HPLC分析图谱。
图15.M5催化的还原胺化产物手性HPLC分析图谱。
图16.M5催化的还原胺化产物手性HPLC分析图谱。
图17.M5催化的还原胺化产物手性HPLC分析图谱。
图18.M5催化的还原胺化产物手性HPLC分析图谱。
图19.M5催化的还原胺化产物手性HPLC分析图谱。
图20.M5催化的还原胺化产物手性HPLC分析图谱。
图21.M5催化的还原胺化产物手性HPLC分析图谱。
图22.M5催化的还原胺化产物手性HPLC分析图谱。
图23.M5催化的还原胺化产物手性HPLC分析图谱。
图24.M5催化的还原胺化产物手性HPLC分析图谱。
图25.M5催化的还原胺化产物手性HPLC分析图谱。
具体实施方式
实施例1
1基于IR-G36蛋白质结构的理性设计
亚胺还原酶IR-G36以下简称IR-G36，其具有序列表中SEQ ID NO.1所述的DNA序列和SEQ ID NO.2所述的氨基酸序列，其与辅因子NADP(H)的共结晶结果显示其结构为经典的同源二聚体。两个单体分别由N端的Rossman结构域和C端的螺旋束构成，中间由一个长的α螺旋链接。在两个单体C端和N端结构域的交界处分别形成两个催化活性中心，并且辅因子NADP(H)结合于两个活性口袋中。
IR-G36活性口袋中距离亚胺中间体
Figure RE-GDA0003659649040000051
范围内的氨基酸共20个，分别为A40、L95、N121、D196、L197、L199、L200、M203、Y204、 F207、W234、M238、V259、S260、N261、M264、Q265、A267、G268和N271。
依据N121的同源位点保守氨基酸N、S、T和A，构建了N121A、 N121T和N121S三个突变体。其中，突变体N121T表现出明显的活性提升，在5mM酮供体浓度下，转化率达到77％，相对于野生型增加了约10倍。同时，突变体N121T的立体选择性也有提升，ee值达到了 88％(R)。
对IR-G36蛋白质的loop252-261上距离辅因子NADPH最近的残基S260作为目标位点进行改造，破坏S260与NADPH的氢键相互作用，从而扩宽底物通道。将S260分别突变为同源位点上的三个保守氨基酸A、N和F，构建突变体。筛选结果显示突变体S260F，在5mM 酮供体浓度下，转化率达到56％，ee值达到了98％(R)。随后，组合突变体M1(N121T/S260F)活性筛选显示，在100mL转化体系中，5 mM酮供体浓度，转化率达到79％，ee值达到了>99％(R)。
2.定点饱和突变
距亚胺中间体
Figure RE-GDA0003659649040000061
以内的20个氨基酸中有2个氨基酸位点高度保守，分别为D196和L200，不利于氨基酸的替换和突变。此外，排除M1中已突变的两个位点，而其余的16个非保守性氨基酸分别为作为目标突变位点。通过单位点的饱和突变，在M1构建16个突变体库，并利用96孔深孔板进行活性筛选，共筛选约1600个突变体。最终，如表1所示，获得18个优势突变体，其活性均高于M1。
表1定点饱和突变筛选获得的优势突变体
Figure RE-GDA0003659649040000071
3.活性中心迭代组合突变
首先将7个位点按照空间距离的远近分为三组，A组(F207和 M238)，B组(L95、M264和A267)，C组(L197和N271)。然后，利用定点饱和突变筛选获得的优势突变，在M1的基础上进行迭代组合突变。
通过对A组进行活性筛选，获得突变体M2 (N121T/S260F/F207I/M238A)具有最高的催化活性。相对于野生型而言，M2的催化效率提高了318倍，Kcat/Km为0.319min-1mM-1。在100mL转化体系中，5g/L酮供体1，转化率达到99％，ee值为>99％ (R)。
第二轮的组合突变以M2为基础，对B组三个氨基酸位点进行组合突变，获得了最优突变体M3 (N121T/S260F/F207I/M238A/M264H/A267H)。相对于野生型而言， M3的催化效率提高了1863倍，Kcat/Km为1.864min-1mM-1。在100mL 转化体系中，15g/L酮供体1，转化率达到97％，ee值为>99％(R)。
第三轮的组合突变以M3为基础，对C组两个氨基酸位点进行组合突变，获得了最优突变体M4 (N121T/S260F/F207I/M238A/M264H/A267H/L197I/N271K)。相对于野生型而言，M4的催化效率提高了3348倍，Kcat/Km为3.349min-1mM-1。在100mL转化体系中，24.9g/L酮供体1，转化率达到96％，ee值为>99％(R)。
4.热稳定性改造
依据‘Consensus’概念(M.Lehmann,et al.,Protein Eng. 2002,15,403–411)，在M4基础上将H169突变为比较保守的Pro 和Ala，构建两个突变体。其中M4-H169A热稳定性明显提高，Tm值达到57.4℃，提高了5.4℃。
在M4的基础上将G145突变为12个极性氨基酸，加强其与周围二级结构的相互作用。经过热稳定性筛选，得到了突变体M5 (N121T/S260F/F207I/M238A/M264H/A267H/L197I/N271K/H169A/G14 5K)，其具有序列表中SEQ ID NO.3所述的DNA序列和SEQ ID NO.4所述的氨基酸序列，其热稳定性明显提高，Tm值达到63.5℃。
此外，M5催化活性也有明显提高，催化效率相对于野生型提高了 4192倍，Kcat/Km值达到4.194min-1mM-1。随后，我们对突变体M5进行了催化活性测试，100mL体系中，24.9g/L的酮供体1浓度，其转化率达到>99％，ee值为>99％。
表2突变体M1-M5的突变位点
Figure RE-GDA0003659649040000091
表3 IR-G36野生型突变体M1-M5的动力学参数和Tm值
Figure RE-GDA0003659649040000092
实施例2
将携带M5表达基因的pET28a质粒用于转化大肠杆菌BL21(DE3) 感受态细胞以进行基因表达。挑取单克隆于含有50μg/mL卡那霉素的LB培养基(10mL)中，37℃，220rpm条件下培养过夜。将上述菌液体按照1：100的接种量，接种于50μg/mL卡那霉素的 1L的LB培养基中，37℃，220rpm条件下培养至OD600值为0.6- 0.8。在培养液中添加IPTG(0.15mM/L)诱导基因表达，并在18℃、 180rpm下继续培养16小时。然后通过5000rpm离心10分钟来收获菌体，并重新悬浮于磷酸钠缓冲液(100mM/L,pH 7.0)中，再经过5000rpm离心10分钟，收集菌体。上述菌体用磷酸钠缓冲液洗涤两次之后，重新悬浮于磷酸钠缓冲液(100mM/L,pH 7.0)中，利用高压破碎仪进行细胞破碎。破碎液以1,2000rpm离心，收集上清液，于真空冷冻干燥器中进行冻干，获得M5的冻干粉。
实施例3
100mL反应体系中，加入酮(1当量)、苄胺(1.1当量)、M5的冻干裂解物(20.9％w/w，M5的冻干裂解物与对应酮的质量比)、D- 葡萄糖(1.5当量，D-葡萄糖的物质的量是酮的1.5倍)、NADP+(1 mM/L)、GDH酶粉(1.5mg/mL)、磷酸钠缓冲液(100mM/L,pH 7.0) 和20％(v/v，DMSO的体积比反应总体积)DMSO，溶液用1M盐酸调节至pH 7.0，将混合液在30℃，220rpm转速下进行反应。
24小时后，反应用乙酸淬灭，得到pH 3.0的溶液，加入硅藻土。过滤混合物并用水冲洗。滤液用二氯甲烷(100mL×3)萃取以除去中性物质。水相用饱和碳酸钠碱化至pH10，然后用二氯甲烷(100 mL×3)萃取。减压除去合并的有机萃取物的溶剂，获得产物。
表4 M5催化的100mL反应体系
Figure RE-GDA0003659649040000101
Figure RE-GDA0003659649040000111
Figure RE-GDA0003659649040000121
实施例4
(R)-3-(苄基氨基)哌啶-1-羧酸叔丁酯(1a)的酶催化合成
Figure RE-GDA0003659649040000122
100mL反应体系中，加入Boc-3-哌啶酮(1)(125mM/L，24.91 当量)、苄胺a(137.5mM/L，是酮当量的1.1倍)、M5的冻干裂解物 (20.9％w/w，是与酮的质量比)、D-葡萄糖(是酮当量的1.5倍)、 NADP+(1mM/L)、GDH酶粉(1.5mg/mL)、磷酸钠缓冲液(100mM/L, pH7.0)和20％(v/v，是与反应液总体积的比)DMSO，溶液用1M 盐酸调节至pH 7.0，将混合液在30℃，220rpm转速下进行反应。
24小时后，反应用乙酸淬灭，得到pH 3.0的溶液，加入硅藻土。过滤混合物并用水冲洗。滤液用二氯甲烷(100mL×3)萃取以除去中性物质。水相用饱和碳酸钠碱化至pH10，然后用二氯甲烷(100 mL×3)萃取。
减压除去合并的有机萃取物的溶剂，得到的(R)-3-(苄基氨基)哌啶-1-羧酸叔丁酯(1a)，浅黄色油(>99.9％e.e.)，如图1。
1H NMRδH(500MHz,CD3OD)7.31-7.37(4H,overlap),7.25 (1H,m),4.06(1H,m),3.76-3.83(3H,overlap),2.85(1H,m), 2.70(1H,m),2.52(1H,m),1.98(1H,m),1.69(1H,m),1.45 (9H,s),1.37(2H,m)。
13C NMRδC(125MHz,CD3OD)156.5,140.7,129.5,129.5, 128.2,81.1,54.0,51.4,31.7,28.7,24.8.
实施例5
(R)-3-(苄基氨基)吡咯烷-1-羧酸叔丁酯(2a)的酶催化合成
Figure RE-GDA0003659649040000131
按照实施例3和4的方法，以Boc-3-吡咯酮和苄胺为原料得到无色油状(48％e.e.)，如图2。
1H NMRδH(500MHz,CD3OD)7.40-7.48(5H,overlap),4.13 (2H,m),3.75(1H,m),3.69(1H,m),3.54(1H,m),3.38(m, 1H),3.38(2H,m),2.31(1H,m),2.05(1H,m),1.46(9H,s)。
13C NMRδC(125MHz,CD3OD)156.1,136.3,136.1,130.3, 130.0,129.6,81.3,58.0,57.2,52.1,50.8,50.3,49.5,49.3, 49.2,49.0,48.8,48.7,48.5,45.3,44.9,30.7,30.0,28.7, 28.7。
实施例6
(R)-3-(苄基氨基)氮杂环庚烷-1-羧酸叔丁酯(3a)的酶催化合成
Figure RE-GDA0003659649040000141
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和苄胺为原料得到无色油状(>99％e.e.)，如图3。
1H NMRδH(500MHz,CD3OD)7.45-7.54(5H,overlap),4.36 (1H,d,J＝12.6Hz),4.22(1H,d,J＝12.6Hz),3.61-3.79 (3H,overlap)3.41(1H,m),3.24(1H,m),2.21(1H,m),1.89(2H,m),1.52-1.66(3H,overlap),1.50(9H,s)。
13C NMRδC(125MHz,CD3OD)158.4,133.7,130.8,130.4, 130.3,130.1,59.5,50.8,49.8,31.7,29.7,28.6,23.4。
实施例7
(R)-4-(苄基氨基)氮杂环庚烷-1-羧酸叔丁酯(4a)的酶催化合成
Figure RE-GDA0003659649040000142
按照实施例3和4的方法，以Boc-4-氮杂环庚烷酮和苄胺为原料得到无色油状(43％e.e.)，如图4。
1H NMRδH(500MHz,CD3OD)7.44-7.52(5H,overlap),4.25 (2H,m),3.65(1H,m),3.46(1H,m),3.41(1H,m),3.31(1H, m),3.21(1H,m),2.34(1H,m),2.23(1H,m),1.99(1H,m), 1.78(1H,m),1.66(2H,m),1.47(9H,s)。
13C NMRδC(125MHz,CD3OD)157.1,157.0,132.8,130.9, 130.6,130.3,81.38,8.3,59.5,59.4,49.9,49.7,47.39,46.7, 43.6,43.0,32.4,32.1,30.6,30.3,28.7,25.6,25.4。
实施例8
(R)-3-(甲基氨基)哌啶-1-羧酸叔丁酯(1b)的酶催化合成
Figure RE-GDA0003659649040000151
按照实施例3和4的方法，以Boc-3-哌啶酮和甲胺为原料得到无色油状(92％e.e.)，如图5。
1H NMRδH(500MHz,CD3OD)4.03(1H,m),3.80(1H,m), 2.87(1H,ddd,J＝13.2,10.9,3.2Hz),2.41(1H,m),2.39 (3H,s),1.99(1H,m),1.70(1H,m),1.70(1H,m),1.46(9H, s),1.44(1H,overlap),1.30(1H,m)。
13C NMRδC(125MHz,CD3OD)156.5,81.1,56.7,33.3,31.4, 28.7。
实施例9
(R)-3-(炔丙基氨基)哌啶-1-羧酸叔丁酯(1c)的酶催化合成
Figure RE-GDA0003659649040000161
按照实施例3和4的方法，以Boc-3-哌啶酮和炔丙胺为原料得到棕色油状(56％e.e.)，如图6。
1H NMRδH(500MHz,CD3OD)4.02(1H,m),3.78(1H,m), 3.53(2H,d,J＝2.6Hz),2.92(1H,m),2.85(1H,m),2.77 (1H,m),2.72(1H,overlap),2.00(1H,m),1.73(1H,m),1.48(1H,overlap),1.46(9H,s),1.37(1H,m)。
13C NMRδC(125MHz,CD3OD)156.5,81.3,74.2,53.5,35.6, 30.8,28.7,24.4。
实施例10
(R)-3-(正丙基氨基)哌啶-1-羧酸叔丁酯(1d)的酶催化合成
Figure RE-GDA0003659649040000162
按照实施例3和4的方法，以Boc-3-哌啶酮和正丙胺为原料得到无色油状(93％e.e.)，如图7。
1H NMRδH(500MHz,CD3OD)4.11(1H,m),3.78(1H,m), 3.18(1H,m),2.99-3.07(4H,overlap),2.16(1H,m),1.80 (1H,m),1.72(2H,m),1.64(1H,m),1.55(1H,m),1.47(9H, s),1.03(3H,t,J＝7.4Hz)。
13C NMRδC(125MHz,CD3OD)156.2,81.9,58.3,54.6,47.9, 28.6,27.9,20.8,18.4,11.3。
实施例11
(R)-3-(正戊基氨基)哌啶-1-羧酸叔丁酯(1e)的酶催化合成
Figure RE-GDA0003659649040000171
按照实施例3和4的方法，以Boc-3-哌啶酮和正戊胺为原料得到无色油状(98％e.e.)，如图8。
1H NMRδH(500MHz,CD3OD)4.10(1H,m),3.77(1H,m), 3.18(1H,m),3.02-3.07(4H,overlap),2.15(1H,m),1.80 (1H,m),1.70(2H,m),1.64(1H,m),1.54(1H,m),1.47(9H, s),1.38-1.44(4H,overlap),0.96(3H,t,J＝7.4Hz)。
13C NMRδC(125MHz,CD3OD)156.2,81.9,54.7,46.4,29.8, 28.8,28.6,28.5,27.9,27.0,23.2,14.1。
实施例12
(R)-3-(环丙基氨基)哌啶-1-羧酸叔丁酯(1f)的酶催化合成
Figure RE-GDA0003659649040000172
按照实施例3和4的方法，以Boc-3-哌啶酮和环丙基胺为原料得到无色油状(87％e.e.)，如图9。
1H NMRδH(500MHz,CD3OD)4.08(1H,m),3.71(1H,m), 3.20(2H,m),3.10(1H,m),2.66(1H,m),2.13(1H,m),1.76 (1H,m),1.64(1H,m),1.57(1H,m),1.47(9H,s),0.79-0.86(4H,m)。
13C NMRδC(125MHz,CD3OD)156.3,81.8,55.8,29.1,28.8, 28.6,23.7,4.8,4.4。
实施例13
(R)-3-(环丁基氨基)哌啶-1-羧酸叔丁酯(1g)的酶催化合成
Figure RE-GDA0003659649040000181
按照实施例3和4的方法，以Boc-3-哌啶酮和环丁基胺为原料得到无色油状(94％e.e.)，如图10。
1H NMRδH(500MHz,CD3OD)3.98(1H,m),3.88(1H,m), 3.70(1H,m),3.07-3.15(3H,overlap),2.34(2H,m),2.22 (2H,m),2.08(1H,m),1.93(2H,m),1.76(1H,m),1.64(1H, m),1.55(1H,m),1.47(9H,s)。
13C NMRδC(125MHz,CD3OD)156.2,81.9,53.1,51.2,28.6, 28.3,28.2,23.6,15.9。
实施例14
(R)-3-(环戊基氨基)哌啶-1-羧酸叔丁酯(1h)的酶催化合成
Figure RE-GDA0003659649040000191
按照实施例3和4的方法，以Boc-3-哌啶酮和环戊基胺为原料得到无色油状(>99％e.e.)，如图11。
1H NMRδH(500MHz,CD3OD)4.16(1H,m),3.81(1H,m), 3.70(1H,m),3.21(1H,m),3.01(2H,m),2.11-2.19(3H, overlap),1.78-1.85(3H,overlap),1.57-1.71(6H,overlap), 1.47(9H,s).
13C NMRδC(125MHz,CD3OD)156.2,82.0,57.6,53.5,30.8, 30.7,28.6,28.3,24.9,24.9.
实施例15
(R)-3-(环己基氨基)哌啶-1-羧酸叔丁酯(1i)的酶催化合成
Figure RE-GDA0003659649040000192
按照实施例3和4的方法，以Boc-3-哌啶酮和环己基胺为原料得到无色油状(98％e.e.)，如图12。
1H NMRδH(500MHz,CD3OD)4.18(1H,m),3.88(1H,m), 3.30(1H,m),3.22(1H,m),2.94(2H,m),2.09-2.16(3H, overlap),1.89(2H,m),1.81(1H,m),1.74(1H,m),1.58(2H,m),1.47(9H,s),1.33-1.42(4H,overlap),1.24(1H,m).
13C NMRδC(125MHz,CD3OD)156.1,82.0,55.4,51.1,30.6, 30.5,28.6,28.4,26.1,25.5,24.1.
实施例16
(R)-3-(2-噻吩乙氨基)哌啶-1-羧酸叔丁酯(1j)的酶催化合成
Figure RE-GDA0003659649040000201
按照实施例3和4的方法，以Boc-3-哌啶酮和2-噻吩乙胺为原料得到黄色油状(>99％e.e.)，如图13。
1H NMRδH(500MHz,CD3OD)7.30(1H,m),6.99(2H, overlap),4.10(1H,m),3.75(1H,m),3.20-3.26(4H,overlap), 3.30-3.35(2H,m),3.05(1H,m),2.16(1H,m),1.80(1H,m),1.67(1H,m),1.55(1H,m),1.46(9H,s)。
13C NMRδC(125MHz,CD3OD)156.1,139.4,128.3,127.3, 125.7,81.9,54.8,47.5,28.6,27.6。
实施例17
(R)-3-(苯乙氨基)哌啶-1-羧酸叔丁酯(1k)的酶催化合成
Figure RE-GDA0003659649040000202
按照实施例3和4的方法，以Boc-3-哌啶酮和苯乙胺为原料得到黄色油状(>99％e.e.)，如图14。
1H NMRδH(500MHz,CD3OD)7.27-7.36(5H,overlap),4.09 (1H,m),3.75(1H,m),3.27(2H,m),3.21(1H,m),2.99-3.08 (4H,overlap),2.16(1H,m),1.80(1H,m),1.66(1H,m),1.55 (1H,m),1.46(9H,s)。
13C NMRδC(125MHz,CD3OD)156.1,137.9,130.0,129.8, 128.3,81.9,54.7,47.5,33.5,28.7,28.6,27.9。
实施例18
(R)-3-(苯丙氨基)哌啶-1-羧酸叔丁酯(1l)的酶催化合成
Figure RE-GDA0003659649040000211
按照实施例3和4的方法，以Boc-3-哌啶酮和苯丙胺为原料得到无色油状(>99％e.e.)，如图15。
1H NMRδH(500MHz,CD3OD)7.18-7.31(overlap,5H),4.05 (1H,m),3.81(1H,m),2.94(1H,m),2.83(4H,overlap),2.71 (2H,t,J＝7.6Hz),2.04(1H,m),1.92(2H,m),1.74(1H, m),1.47(2H,overlap),1.47(9H,s)。
13C NMRδC(125MHz,CD3OD)156.4,142.5,129.5,129.4, 127.2,81.5,55.0,46.8,34.2,31.1,30.2,28.6,24.4。
实施例19
(R)-3-[2-(吲哚-3-基)乙基氨基]哌啶-1-羧酸叔丁酯(1m)的酶催化合成
Figure RE-GDA0003659649040000221
按照实施例3和4的方法，以Boc-3-哌啶酮和2-(吲哚-3-基)乙胺为原料得到黄色油状(95％e.e.)，如图16。
1H NMRδH(500MHz,CD3OD)7.58(1H,dd,J＝7.9,1.1 Hz),7.37(1H,dd,J＝8.2,1.1Hz),7.18(1H,s),7.13(1H, ddd,J＝8.2,6.9,1.1Hz),7.05(1H,ddd,J＝7.9,6.9,1.1Hz),4.10(1H,m),3.75(1H,m),3.35(2H,m),3.15-3.24(4H, m),3.04(1H,m),2.14(1H,m),1.78(1H,m),1.64(1H,m), 1.53(1H,m),1.45(9H,s)。
13C NMRδC(125MHz,CD3OD)156.2,138.3,128.1,124.1, 122.8,120.1,118.9,112.6,110.3,81.9,54.6,46.8,28.6, 23.5。
实施例20
(R)-3-(甲基氨基)氮杂环庚烷-1-羧酸叔丁酯(3b)的酶催化合成
Figure RE-GDA0003659649040000231
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和甲胺为原料得到无色油状(95％e.e.)，如图17。
1H NMRδH(500MHz,CD3OD)3.74(1H,dd,J＝15.4,4.8 Hz),3.68(1H,m),3.59(1H,dd,J＝15.4,5.0Hz),3.29(1H, m),3.17(1H,m),2.18(1H,m),1.85(2H,m),1.65(1H,m),1.50(2H,m),1.49(9H,s)。
13C NMRδC(125MHz,CD3OD)158.3,82.0,61.0,50.0,48.4, 32.1,30.9,29.9,28.6,23.1。
实施例21
(R)-3-(炔丙基氨基)氮杂环庚烷-1-羧酸叔丁酯(3c)的酶催化合成
Figure RE-GDA0003659649040000232
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和炔丙基胺为原料得到棕色油状(98％e.e.)，如图18。
1H NMRδH(500MHz,CDCl3)3.69(2H,m),3.58(0.5H,dd, J＝14.5,4.7Hz),3.53(1H,m),3.44(1H,m),3.25(1H,m), 3.14(1H,dt,J＝13.5,5.4Hz),3.01(0.5H,m),2.92(1H, dd,J＝14.1,9.0Hz),2.31(0.5H,s),2.27(0.5H,s),1.99 (0.5H,m),1.73-1.88(2.5H,overlap),1.59(1H,m),1.48 (4.5H,s),1.45(4.5H,s),1.39(2H,m)。
13C NMRδC(125MHz,CDCl3)156.6,155.5,80.7,80.2, 79.9,78.8,73.9,72.6,56.8,56.7,49.5,49.4,48.8,46.9, 35.5,35.4,34.3,32.1,28.6,28.5,27.5,22.6,22.5。
实施例22
(R)-3-(正丙基氨基)氮杂环庚烷-1-羧酸叔丁酯(3d)的酶催化合成
Figure RE-GDA0003659649040000241
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和正丙基胺为原料得到无色油状。
1H NMRδH(500MHz,CD3OD)3.62-3.69(3H,m),3.37(1H, m),3.25(1H,m),3.11(1H,m),3.03(1H,m),2.22(1H,m), 1.89(2H,m),1.75(2H,m),1.65(1H,m),1.51(9H,s),1.47(2H,m),1.06(3H,t,J＝7.4Hz)。
13C NMRδC(125MHz,CD3OD)156.9,80.7,58.2,48.4,47.3, 47.0,29.8,28.3,27.2,21.9,19.5,9.8。
实施例23
(R)-3-(正戊基氨基)氮杂环庚烷-1-羧酸叔丁酯(3e)的酶催化合成
Figure RE-GDA0003659649040000251
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和正戊基胺为原料得到无色油状。
1H NMRδH(500MHz,CD3OD)3.61-3.66(3H,overlap),3.34 (1H,m),3.22(1H,m),3.11(1H,m),3.02(1H,m),2.18(1H, m),1.88(2H,m),1.69(3H,overlap),1.49(9H,s),1.40(6H, overlap),0.96(3H,t,J＝7.4Hz)。
13C NMRδC(125MHz,CD3OD)158.3,82.1,59.6,49.8,48.4, 47.2,31.3,29.7,29.7,28.6,27.2,23.3,23.2,14.1。
实施例24
(R)-3-(环丙基氨基)氮杂环庚烷-1-羧酸叔丁酯(3f)的酶催化合成
Figure RE-GDA0003659649040000252
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和环丙胺为原料得到无色油状(87％e.e.)，如图19。
1H NMRδH(500MHz,CDCl3)3.97(0.5H,d,J＝12.4Hz), 3.71(1H,m),3.49(1H,m),3.32(0.5H,m),3.24(0.5H,m), 3.13(0.5H,m),2.99(1H,m),2.46(0.5H,m),2.29(0.5H,m),2.18(0.5H,m),1.98(0.5H,m),1.86(1H,m),1.78(1H,m), 1.57(1H,m),1.47(4.5H,s),1.46(4.5H,s),1.38(2H,m), 0.84(1H,m),0.65(3H,m)。
13C NMRδC(125MHz,CDCl3)156.9,155.5,80.5,79.8, 59.1,58.9,49.7,49.6,49.3,47.0,34.0,31.5,28.8,28.75, 28.7,28.6,28.5,27.6,22.5,22.45,6.3,5.3,5.2,4.8。
实施例25
(R)-3-(环丁基氨基)氮杂环庚烷-1-羧酸叔丁酯(3g)的酶催化合成
Figure RE-GDA0003659649040000261
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和环丁胺为原料得到无色油状(89％e.e.)，如图20。
1H NMRδH(500MHz,CD3DOCD3)3.84(0.5H,m),3.78(0.5H, m),3.69(1H,m),3.55(0.5H,m),3.43(0.5H,m),3.32(0.5H, dt,J＝13.5,5.5Hz),3.24(0.5H,dd,J＝13.9,6.9Hz), 3.11(0.5H,m),3.02(0.5H,m),2.94(1H,m),2.23(2H,m), 2.07(1H,m),1.95(1.5H,m),1.67-1.85(4.5H,overlap),1.58 (1H,m),1.50(1H,m),1.47(4.5H,s),1.45(4.5H,s),1.58 (1H,m)。
13C NMRδC(125MHz,CD3DOCD3)156.6,155.5,79.8,79.5, 56.8,52.4,52.0,50.9,50.3,48.9,47.5,34.3,32.7,30.9, 30.8,30.1,29.9,29.0,28.6,28.5,28.2,23.2,23.0,15.5, 15.45。
实施例26
(R)-3-(环戊基氨基)氮杂环庚烷-1-羧酸叔丁酯(3h)的酶催化合成
Figure RE-GDA0003659649040000271
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和环戊胺为原料得到无色油状(96％e.e.)，如图21。
1H NMRδH(500MHz,CDCl3)4.07(0.5H,m),3.86(1H,m), 3.54(0.5H,m),3.41(1.5H,overlap),3.32(1H,m),3.11(1H, m),2.98(0.5H,m),2.02-2.19(3H,overlap),1.57-1.86(10H, overlap),1.47(4.5H,s),1.45(4.5H,s),1.36(1H,m)。
13C NMRδC(125MHz,CDCl3)156.5,155.2,80.5,80.3, 56.9,56.7,56.3,56.26,48.5,48.0,47.9,47.0,32.1,31.2, 30.5,30.4,30.0,29.7,28.6,28.5,28.3,27.3,24.0,24.0, 23.9,22.4,22.2。
实施例27
(R)-3-(环己基氨基)氮杂环庚烷-1-羧酸叔丁酯(3i)的酶催化合成
Figure RE-GDA0003659649040000281
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和环己胺为原料得到无色油状。
1H NMRδH(500MHz,CD3DOCD3)3.97(0.5H,m),3.82(1H, m),3.45(2H,m),3.22(0.5H,m),3.09(1.5H,overlap),2.92 (0.5H,m),2.08(1H,m),1.85(4.5H,overlap),1.69(3.5H, overlap),1.55(4.5H,s),1.53(4.5H,s),1.24-1.42(7H, overlap)。
13C NMRδC(125MHz,CD3DOCD3)156.9,155.6,80.1,79.6, 54.8,54.7,54.4,50.7,50.0,48.9,47.3,34.1,32.9,32.5, 32.3,32.0,31.3,29.0,28.7,28.5,28.0,26.5,26.3,25.6, 25.5,25.45,25.4,23.1,22.9。
实施例28
(R)-3-(2-噻吩乙氨基)氮杂环庚烷-1-羧酸叔丁酯(3j)的酶催化合成
Figure RE-GDA0003659649040000291
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和2-噻吩乙胺为原料得到黄色油状(93％e.e.)，如图22。
1H NMRδH(500MHz,CD3OD)7.31(1H,dd,J＝5.1,1.4 Hz),6.99(2H,overlap),3.60-3.72(3H,m),3.40(2H,m), 3.26(3H,overlap),3.18(1H,m),2.21(1H,m),1.90(1H,m),1.81(1H,m),1.63(1H,m),1.47(9H,s),1.44(2H,m)。
13C NMRδC(125MHz,CD3OD)158.4,139.3,128.4,127.5, 125.9,82.1,59.8,50.0,48.6,48.3,31.2,29.8,28.6,27.7, 23.2。
实施例29
(R)-3-(苯乙基氨基)氮杂环庚烷-1-羧酸叔丁酯(3k)的酶催化合成
Figure RE-GDA0003659649040000292
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和苯乙胺为原料得到无色油状(98％e.e.)，如图23。
1H NMRδH(500MHz,CD3OD)7.26-7.36(5H,overlap),3.60- 3.73(3H,overlap),3.40(2H,m),3.29(1H,m),3.17(1H,m), 3.01(2H,m),2.21(1H,m),1.90(1H,m),1.81(1H,m),1.63 (1H,m),1.46(9H,s),1.43(2H,overlap)。
13C NMRδC(125MHz,CD3OD)158.4,137.7,130.0,129.8, 128.3,82.1,59.8,50.0,48.9,48.3,33.6,31.2,28.6,28.6, 23.2。
实施例30
(R)-3-(苯丙基氨基)氮杂环庚烷-1-羧酸叔丁酯(3l)的酶催化合成
Figure RE-GDA0003659649040000301
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和苯丙胺为原料得到无色油状(94％e.e.)，如图24。
1H NMRδH(500MHz,CD3OD)7.30(2H,overlap),7.23(3H, overlap),3.64(2H,m),3.35(1H,m),3.22(2H,m),3.14(1H, m),3.05(1H,m),2.74(2H,m),1.99-2.18(3H,m),1.88(2H, m),1.61(1H,m),1.49(9H,s),1.44(2H,m)。
13C NMRδC(125MHz,CD3OD)158.3,141.6,129.7,129.4, 127.5,82.1,59.6,49.8,48.3,46.7,33.5,31.2,29.7,29.2, 28.6,23.2。
实施例31
(R)-3-[2-(吲哚-3-基)乙基氨基]氮杂环庚烷-1-羧酸叔丁酯(3m) 的酶催化合成
Figure RE-GDA0003659649040000311
按照实施例3和4的方法，以Boc-3-氮杂环庚烷酮和2-(吲哚- 3-基)乙胺为原料得到黄色油状(>99％e.e.)，如图25。
1H NMRδH(500MHz,CD3OD)7.60(1H,d,J＝8.0Hz),7.37 (1H,d,J＝8.0Hz),7.21(1H,s),7.12(1H,dd),7.05(1H, dd),3.53-3.71(3H,m),3.33-3.45(3H,m),3.18(2H,t,J＝7.1Hz),3.09(1H,ddd,J＝14.4,10.6,4.4Hz),2.21(1H, m),1.88(1H,m),1.78(1H,m),1.57(1H,m),1.41(2H, overlap),1.40(9H,s)。
13C NMRδC(125MHz,CD3OD)158.5,138.4,128.1,124.5, 122.8,120.1,118.9,112.6,109.9,82.1,59.7,50.1,48.7, 47.5,31.1,30.0,28.5,23.6,23.2。
实施例32
使用M5进行(R)-3-(苄基氨基)哌啶-1-羧酸叔丁酯(1a)的制备级生物转化
Figure RE-GDA0003659649040000321
首先，将0.8L DMSO和3.2L磷酸钠缓冲液(100mM,pH 7.0) 加入5L生物反应器中，并搅拌。
然后，将苄胺a(137.5mM/L，1.1当量)、D-葡萄糖(1.5倍酮的物质的量)和烟酰胺腺嘌呤二核苷酸磷酸二钠盐(NADP+)(3.1g) 的混合液加入反应器中，在30℃条件下搅拌。
再将亚胺还原酶突变体M5的冻干酶粉(20.8g，20.9％w/w，与酮的质量比)和GDH酶粉末(4.0g，4％w/w，与酮的质量比)添加到反应溶液中，并搅拌。最后，向反应器中加入N-Boc-3-哌啶酮1 (125mM/L，1当量)，并使用HPLC分析监测反应。24小时后，转化率达到97.4％。随后，用乙酸淬灭反应并搅拌以得到pH 3.0的溶液。将硅藻土(100g)装入生物反应器并搅拌10分钟。过滤混合物并用水冲洗。滤液用二氯甲烷(4L×3)萃取以除去中性物质。用饱和碳酸钠将水相碱化至pH 10，然后用二氯甲烷(4L×3)萃取。减压除去合并的有机萃取物的溶剂，得到黄色油(R)-3-(苄基氨基)哌啶-1-羧酸叔丁酯(81％分离产率，>99％e.e.)。
本发明通过合理的酶空腔设计和热稳定性工程对天然酶进行了改造得到了突变体M5，通过一系列手性氮杂环烷基胺的合成证明了其高选择性、高活性、高立体选择性、高转化率以及优秀的底物相容性。
序列表
<110> 江苏集萃分子工程研究院有限公司
<120> 亚胺还原酶突变体M5及其在合成氮杂环手性胺中的应用
<130> P22007
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人工序列(Artificial Sequence)
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atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgccggaat ctaccacccc gagtaccgcc accccggtga ccatcatcgg tcttggtgca 120
atgggcaccg ccctggcaaa cgcattcctc gatgcaggtc atagtaccac cgtttggaat 180
cgtaccgcag cacgcgccac cgcattagcc gcacgcggcg cacatcatgc agaaaccgtg 240
accgaagcca ttgcagcctc tccgttagtg attgcctgtg tgctggatta tgatgccttt 300
catgaaacct tagccccggc tacagacgcg ctggcaggtc gcgccctggt taatctgacc 360
aatggtaccc cgaaacaggc acgcgaaacc gcctcttggg cagccgatca tcgtattgat 420
tatctggatg gcggtattat ggccattccg ccgggtattg caaccccgga tagttttatt 480
ctgtatagcg gtccgttagg tacctttgaa gcacatcgct caaccttaga agtgctgggc 540
gcagcaaatc atgtgggtac cgatcatggt ttggcgagct tacatgattt agcactgctg 600
accggtatgt atggcatgtt tgcaggcatt ttacaggcct ttgccttaat tgatagtgaa 660
ggtattccgg caggcgatct ggccccgatg ttaaccaatt ggttaaccgg catggcacat 720
agcgtggccc attatgccca gcagattgat accggcgatt atgaaaccgg tgttgtgtct 780
aatttagcaa tgcagagcgc cggctttgca aatttagttc aggccggtga agatcagggt 840
gtggatgtgg gcttactgcg tccgctgttt gaactgatgc gtcatcaggt tgccgcaggc 900
tatggtaatg gtgatgttgc ctcagttatt gaactgattc gtcgcgaaga acgtcgtcag 960
ccggccaaaa gtccgggcgc agataaaatt acccgtgcac gtcgtccgta a 1011
<210> 2
<211> 336
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Pro Glu Ser Thr Thr Pro Ser Thr Ala Thr Pro
20 25 30
Val Thr Ile Ile Gly Leu Gly Ala Met Gly Thr Ala Leu Ala Asn Ala
35 40 45
Phe Leu Asp Ala Gly His Ser Thr Thr Val Trp Asn Arg Thr Ala Ala
50 55 60
Arg Ala Thr Ala Leu Ala Ala Arg Gly Ala His His Ala Glu Thr Val
65 70 75 80
Thr Glu Ala Ile Ala Ala Ser Pro Leu Val Ile Ala Cys Val Leu Asp
85 90 95
Tyr Asp Ala Phe His Glu Thr Leu Ala Pro Ala Thr Asp Ala Leu Ala
100 105 110
Gly Arg Ala Leu Val Asn Leu Thr Asn Gly Thr Pro Lys Gln Ala Arg
115 120 125
Glu Thr Ala Ser Trp Ala Ala Asp His Arg Ile Asp Tyr Leu Asp Gly
130 135 140
Gly Ile Met Ala Ile Pro Pro Gly Ile Ala Thr Pro Asp Ser Phe Ile
145 150 155 160
Leu Tyr Ser Gly Pro Leu Gly Thr Phe Glu Ala His Arg Ser Thr Leu
165 170 175
Glu Val Leu Gly Ala Ala Asn His Val Gly Thr Asp His Gly Leu Ala
180 185 190
Ser Leu His Asp Leu Ala Leu Leu Thr Gly Met Tyr Gly Met Phe Ala
195 200 205
Gly Ile Leu Gln Ala Phe Ala Leu Ile Asp Ser Glu Gly Ile Pro Ala
210 215 220
Gly Asp Leu Ala Pro Met Leu Thr Asn Trp Leu Thr Gly Met Ala His
225 230 235 240
Ser Val Ala His Tyr Ala Gln Gln Ile Asp Thr Gly Asp Tyr Glu Thr
245 250 255
Gly Val Val Ser Asn Leu Ala Met Gln Ser Ala Gly Phe Ala Asn Leu
260 265 270
Val Gln Ala Gly Glu Asp Gln Gly Val Asp Val Gly Leu Leu Arg Pro
275 280 285
Leu Phe Glu Leu Met Arg His Gln Val Ala Ala Gly Tyr Gly Asn Gly
290 295 300
Asp Val Ala Ser Val Ile Glu Leu Ile Arg Arg Glu Glu Arg Arg Gln
305 310 315 320
Pro Ala Lys Ser Pro Gly Ala Asp Lys Ile Thr Arg Ala Arg Arg Pro
325 330 335
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgccggaat ctaccacccc gagtaccgcc accccggtga ccatcatcgg tcttggtgca 120
atgggcaccg ccctggcaaa cgcattcctc gatgcaggtc atagtaccac cgtttggaat 180
cgtaccgcag cacgcgccac cgcattagcc gcacgcggcg cacatcatgc agaaaccgtg 240
accgaagcca ttgcagcctc tccgttagtg attgcctgtg tgctggatta tgatgccttt 300
catgaaacct tagccccggc tacagacgcg ctggcaggtc gcgccctggt taatctgacc 360
acaggtaccc cgaaacaggc acgcgaaacc gcctcttggg cagccgatca tcgtattgat 420
tatctggatg gcaaaattat ggccattccg ccgggtattg caaccccgga tagttttatt 480
ctgtatagcg gtccgttagg tacctttgaa gcacatcgct caaccttaga agtgctgggc 540
gcagcaaatc atgtgggtac cgatgcaggt ttggcgagct tacatgatat tgcactgctg 600
accggtatgt atggcatgat tgcaggcatt ttacaggcct ttgccttaat tgatagtgaa 660
ggtattccgg caggcgatct ggccccgatg ttaaccaatt ggttaaccgg cgcagcacat 720
agcgtggccc attatgccca gcagattgat accggcgatt atgaaaccgg tgttgtgttt 780
aatttagcac atcagagcca tggctttgca aaattagttc aggccggtga agatcagggt 840
gtggatgtgg gcttactgcg tccgctgttt gaactgatgc gtcatcaggt tgccgcaggc 900
tatggtaatg gtgatgttgc ctcagttatt gaactgattc gtcgcgaaga acgtcgtcag 960
ccggccaaaa gtccgggcgc agataaaatt acccgtgcac gtcgtccgta a 1011
<210> 4
<211> 336
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Pro Glu Ser Thr Thr Pro Ser Thr Ala Thr Pro
20 25 30
Val Thr Ile Ile Gly Leu Gly Ala Met Gly Thr Ala Leu Ala Asn Ala
35 40 45
Phe Leu Asp Ala Gly His Ser Thr Thr Val Trp Asn Arg Thr Ala Ala
50 55 60
Arg Ala Thr Ala Leu Ala Ala Arg Gly Ala His His Ala Glu Thr Val
65 70 75 80
Thr Glu Ala Ile Ala Ala Ser Pro Leu Val Ile Ala Cys Val Leu Asp
85 90 95
Tyr Asp Ala Phe His Glu Thr Leu Ala Pro Ala Thr Asp Ala Leu Ala
100 105 110
Gly Arg Ala Leu Val Asn Leu Thr Thr Gly Thr Pro Lys Gln Ala Arg
115 120 125
Glu Thr Ala Ser Trp Ala Ala Asp His Arg Ile Asp Tyr Leu Asp Gly
130 135 140
Lys Ile Met Ala Ile Pro Pro Gly Ile Ala Thr Pro Asp Ser Phe Ile
145 150 155 160
Leu Tyr Ser Gly Pro Leu Gly Thr Phe Glu Ala His Arg Ser Thr Leu
165 170 175
Glu Val Leu Gly Ala Ala Asn His Val Gly Thr Asp Ala Gly Leu Ala
180 185 190
Ser Leu His Asp Ile Ala Leu Leu Thr Gly Met Tyr Gly Met Ile Ala
195 200 205
Gly Ile Leu Gln Ala Phe Ala Leu Ile Asp Ser Glu Gly Ile Pro Ala
210 215 220
Gly Asp Leu Ala Pro Met Leu Thr Asn Trp Leu Thr Gly Ala Ala His
225 230 235 240
Ser Val Ala His Tyr Ala Gln Gln Ile Asp Thr Gly Asp Tyr Glu Thr
245 250 255
Gly Val Val Phe Asn Leu Ala His Gln Ser His Gly Phe Ala Lys Leu
260 265 270
Val Gln Ala Gly Glu Asp Gln Gly Val Asp Val Gly Leu Leu Arg Pro
275 280 285
Leu Phe Glu Leu Met Arg His Gln Val Ala Ala Gly Tyr Gly Asn Gly
290 295 300
Asp Val Ala Ser Val Ile Glu Leu Ile Arg Arg Glu Glu Arg Arg Gln
305 310 315 320
Pro Ala Lys Ser Pro Gly Ala Asp Lys Ile Thr Arg Ala Arg Arg Pro
325 330 335

Claims (10)

1.一种亚胺还原酶突变体M5，其具有序列表中SEQ ID NO.4所述的氨基酸序列。
2.权利要求1所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，
所述酮为
Figure FDA0003561337300000012
其中R1为
Figure FDA0003561337300000013
n为1-10的整数，m为1-10的整数；
所述胺为R2-NH2，其中，R2为
Figure FDA0003561337300000011
X为1-10的整数。
3.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应中加入酮与胺的物质的量比为1:1.1。
4.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应中加入还原胺化酶突变体M5的冻干裂解物，所述还原胺化酶突变体M5的冻干裂解物与酮的质量比为4-20％。
5.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应中加入D-葡萄糖，所述D-葡萄糖与酮的物质的量比为1.5：1。
6.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应中加入NADP+的量为1mM/L；
GDH酶粉的量为1.5mg/mL；
磷酸钠缓冲液的量为100mM/L；DMSO，所述DMSO的体积用量是反应液总体积的20％。
7.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应的溶液pH 7.0。
8.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应的温度为30℃。
9.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应的时间为24小时。
10.依据权利要求2所述还原胺化酶突变体M5在酮与胺合成烷基化胺的反应中的应用，其特征在于，所述反应的步骤包括：
将体积比为1:4的DMSO和磷酸钠缓冲液加入生物反应器中；
将胺、D-葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸二钠盐的混合液加入反应器中，在30℃条件下反应；所述胺为苄胺，所述苄胺与酮的物质的量比为1.1:1；
将亚胺还原酶突变体M5的冻干酶粉和GDH酶粉末添加到反应溶液中反应；所述亚胺还原酶突变体M5的冻干酶粉与酮的质量比为4％；
反应24小时后得产物；所述酮为N-Boc-3-哌啶酮，所述N-Boc-3-哌啶酮的加入量为125mM/L。
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