CN110564788A - 一种利用亚胺还原酶生产麻黄碱的方法 - Google Patents

一种利用亚胺还原酶生产麻黄碱的方法 Download PDF

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CN110564788A
CN110564788A CN201910942604.0A CN201910942604A CN110564788A CN 110564788 A CN110564788 A CN 110564788A CN 201910942604 A CN201910942604 A CN 201910942604A CN 110564788 A CN110564788 A CN 110564788A
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穆晓清
徐岩
罗磊
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Abstract

本发明公开了一种利用亚胺还原酶生产麻黄碱的方法，属于生物技术领域。本发明提供了一种利用亚胺还原酶生产麻黄碱的方法，利用此方法以(R)‑1‑苯基‑2‑羰基‑3‑丙醇与甲胺为反应原料反应18h，即可使反应液中麻黄碱的产率高达86.5％，利用此方法以苯甲醛、丙酮酸与甲胺为反应原料反应18h，即可使反应液中麻黄碱的产率高达62.7％，因此，利用此方法生产麻黄碱具有产量高的优势。

Description

一种利用亚胺还原酶生产麻黄碱的方法
技术领域
本发明涉及一种利用亚胺还原酶生产麻黄碱的方法，属于生物技术领域。
背景技术
中药麻黄具有良好的药用价值，且具有用药安全可靠、耐受性强、副作用小的优点。它的主要有效成分为麻黄碱(ephedrine)和伪麻黄碱。其中，麻黄碱属于拟肾上腺激动药，可用于治疗支气管哮喘、各种咳嗽、过敏反应以及低血压等症；并且，麻黄碱还具有松弛平滑肌、收缩血管、加速心率、升高血压及中枢神经兴奋作用，被应用在许多感冒药中；另外，有研究表明，麻黄碱可作为食品添加剂，兴奋中枢并抑制肠蠕动以控制体重，在节食、减肥中有巨大的应用前景。
目前，对麻黄碱的生产，国内主要采用植物提取法直接从麻黄草中提取麻黄碱，但是，由于麻黄草资源限制，大多数麻黄碱生产企业因原料不足而处于半停产状态，麻黄碱产量不足已严重影响了我国医药企业的发展和麻黄碱类药品在国际市场上的竞争力。国内外也有部分厂家，例如，深圳沃兰德和内蒙古赤峰等，使用化学合成法生产麻黄碱，此方法为先生产L-麻黄碱和D-伪麻黄碱的外消旋体，然后将生产得到的外消旋体进行拆分，但是，此方法所使用的拆分试剂价格昂贵，成本过高。
与植物提取法和化学合成法相比，生物转化法天然具有原料来源广以及生产成本低的优势。为解决麻黄碱生产过程中原料不足以及生产成本高的问题，1921年，Neuberg等人采用酵母转化苯甲醛和丙酮酸生产得到了L-苯基乙酰基甲醇(具体可参考文献：NeubergC,Libermann L.Zur Kenntnis der Carboligase[J].Biochem.Z.,1921,121:311-325.)，随后，Hildebrandt等人继续将L-苯基乙酰基甲醇与甲基胺反应合成了麻黄碱(具体可参考文献：Hildebrandt G,Klavehn W.1-Phenyl-2-methylamino-1-propanol[P].US Pat.:1956930(C.A.-28:4072),1934-05-01.)，这首次实现了麻黄碱的半生物转化。
但是，上述方法依旧存在产量低以及生产得到的麻黄碱杂质过多的缺陷，并不能实现真正的工业化生产。因此，仍需找到一种操作简单、产量高、生产成本低且生产得到的麻黄碱杂质少、稳定性好的生产麻黄碱的方法，以早日实现麻黄碱的工业化生产。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种操作简单、产量高、生产成本低且生产得到的麻黄碱杂质少、稳定性好的生产麻黄碱的方法。
[技术方案]
为解决上述技术问题，本发明提供了一种生产麻黄碱的方法，所述方法为以(R)-1-苯基-2-羰基-3-丙醇与甲胺为反应原料，将(R)-1-苯基-2-羰基-3-丙醇与甲胺在亚胺还原酶或表达亚胺还原酶的基因工程菌的作用下进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱；
或者，所述方法为以苯甲醛、丙酮酸与甲胺为反应原料，将苯甲醛、丙酮酸与甲胺在亚胺还原酶或表达亚胺还原酶的基因工程菌，和，丙酮酸脱羧酶或表达丙酮酸脱羧酶的基因工程菌的共同作用下进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱。
在本发明的一种实施方式中，所述亚胺还原酶为氨基酸序列如SEQ ID No.11、SEQID No.12、SEQ ID No.13或SEQ ID No.14所示的亚胺还原酶中的一种或多种。
在本发明的一种实施方式中，所述丙酮酸脱羧酶为氨基酸序列如SEQ ID No.15或SEQ ID No.16所示的亚胺还原酶中的一种或多种。
在本发明的一种实施方式中，所述方法为以(R)-1-苯基-2-羰基-3-丙醇与甲胺为反应原料，先将(R)-1-苯基-2-羰基-3-丙醇与甲胺加入缓冲液中，得到反应体系，然后在反应体系中加入亚胺还原酶或表达亚胺还原酶的基因工程菌进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱；
或者，所述方法为以苯甲醛、丙酮酸与甲胺为反应原料，先将苯甲醛、丙酮酸与甲胺加入缓冲液中，得到反应体系，然后在反应体系中加入亚胺还原酶或表达亚胺还原酶的基因工程菌，和，丙酮酸脱羧酶或表达丙酮酸脱羧酶的基因工程菌进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱。
在本发明的一种实施方式中，当反应原料为(R)-1-苯基-2-羰基-3-丙醇与甲胺时，所述反应体系中还含有葡萄糖脱氢酶、葡萄糖、第一辅酶以及二甲基亚砜，或者，所述反应体系中还含有表达葡萄糖脱氢酶的基因工程菌、葡萄糖、第一辅酶以及二甲基亚砜；
当反应原料为苯甲醛、丙酮酸与甲胺时，所述反应体系中还含有葡萄糖脱氢酶、葡萄糖、第一辅酶、第二辅酶以及二甲基亚砜，或者，所述反应体系中还含有表达葡萄糖脱氢酶的基因工程菌、葡萄糖、第一辅酶、第二辅酶以及二甲基亚砜。
在本发明的一种实施方式中，所述第一辅酶为NADPH和/或NADP+。
在本发明的一种实施方式中，所述第二辅酶为焦磷酸硫胺素。
在本发明的一种实施方式中，所述反应体系中，亚胺还原酶的浓度为1.2～30U/mL。
在本发明的一种实施方式中，所述反应体系中，可表达亚胺还原酶的基因工程菌的浓度为16～50g/L。
在本发明的一种实施方式中，所述反应体系中，丙酮酸脱羧酶的浓度为2～12U/mL。
在本发明的一种实施方式中，所述反应体系中，可表达丙酮酸脱羧酶的基因工程菌的浓度为20～80g/L。
在本发明的一种实施方式中，所述反应体系中，葡萄糖脱氢酶的浓度为1～8U/mL。
在本发明的一种实施方式中，所述反应体系中，可表达葡萄糖脱氢酶的基因工程菌的浓度为14～56g/L。
在本发明的一种实施方式中，所述反应体系中，葡萄糖的浓度为50～300g/L。
在本发明的一种实施方式中，所述反应体系中，第一辅酶的浓度为0.4～5g/L。
在本发明的一种实施方式中，所述反应体系中，第二辅酶的浓度为0.01～25mmol/L。
在本发明的一种实施方式中，所述反应体系中，二甲基亚砜的浓度为5～20％(v/v)。
在本发明的一种实施方式中，所述反应的温度为20～35℃、pH为6.5～8.5、转速为200～350rpm。
在本发明的一种实施方式中，所述缓冲液为磷酸盐缓冲液、碳酸盐缓冲液、Tri-HCl缓冲液、硼酸盐缓冲液、甘氨酸缓冲液、柠檬酸盐缓冲液或MOPS缓冲液。
在本发明的一种实施方式中，所述表达亚胺还原酶的基因工程菌以大肠杆菌为表达宿主、以质粒pET-28a(+)为表达载体。
在本发明的一种实施方式中，所述表达丙酮酸脱羧酶的基因工程菌以大肠杆菌为表达宿主、以质粒pET-28a(+)为表达载体。
本发明还提供了亚胺还原酶或表达亚胺还原酶的基因工程菌或上述一种生产麻黄碱的方法在生产麻黄碱中的应用。
在本发明的一种实施方式中，所述亚胺还原酶为氨基酸序列如SEQ ID No.11、SEQID No.12、SEQ ID No.13或SEQ ID No.14所示的亚胺还原酶中的一种或多种。
[有益效果]
(1)本发明提供了一种利用亚胺还原酶生产麻黄碱的方法，利用此方法以(R)-1-苯基-2-羰基-3-丙醇与甲胺为反应原料反应18h，即可使反应液中麻黄碱的产率高达86.5％，利用此方法以苯甲醛、丙酮酸与甲胺为反应原料反应18h，即可使反应液中麻黄碱的产率高达62.7％，因此，利用此方法生产麻黄碱具有产量高的优势。
(2)本发明提供了一种利用亚胺还原酶生产麻黄碱的方法，利用此方法以(R)-1-苯基-2-羰基-3-丙醇与甲胺为反应原料生产得到的麻黄碱的光学纯度高达99.6％，利用此方法以苯甲醛、丙酮酸与甲胺为反应原料生产得到的麻黄碱的光学纯度高达92.1％，因此，利用此方法生产得到的麻黄碱杂质含量少、稳定性好。
(3)本发明提供了一种利用亚胺还原酶生产麻黄碱的方法，利用此方法可以(R)-1-苯基-2-羰基-3-丙醇与甲胺，或者，苯甲醛、丙酮酸与甲胺为反应原料生产麻黄碱，这些反应原料易获得且获得成本较低，因此，利用此方法生产麻黄碱具有成本低的优势。
(4)本发明提供了一种利用亚胺还原酶生产麻黄碱的方法，利用此方法生产麻黄碱仅需将相关原料和相关酶或可表达相关酶的相关基因工程菌投入反应体系中即可完成，因此，利用此方法生产麻黄碱具有操作简单的优势。
具体实施方式
以下结合具体实施例对本发明作进一步的详细描述，但该实施例不应该理解为对本发明的限制，仅作举例而已。
下述实施例中涉及的大肠杆菌(Escherichia coli)BL21(DE3)购自北纳生物；下述实施例中涉及的苯甲醛、丙酮酸、甲胺、磷酸盐缓冲液、NADPH、NADP+、焦磷酸硫胺素和二甲基亚砜购自国药集团；述实施例中涉及的葡萄糖脱氢酶购自上海研生生化试剂有限公司。
下述实施例中涉及的培养基如下：
LB液体培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠。
LB固体培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠、20g/L琼脂。
下述实施例中涉及的检测方法如下：
(R)-1-苯基-2-羰基-3-丙醇产率以及光学纯度的检测方法：参考文献“Continuous Production of(R)-Phenylacetylcarbinol in an Enzyme-MembraneReactor Using a Potent Mutant of Pyruvate Decarboxylase from Zymomonasmobilis,(2001).”。
麻黄碱产率以及光学纯度的的检测方法：参考文献“K.Deventer,O.J.Pozo,P.VanEenoo,F.T.Delbeke,Development and validation of an LC-MS/MS method for thequantification of ephedrines in urine,J Chromatogr B Analyt Technol BiomedLife Sci,877(2009)369-374.”。
下述实施例中涉及的可表达丙酮酸脱羧酶的基因工程菌以及(R)-1-苯基-2-羰基-3-丙醇的制备方法如下：
化学合成编码氨基酸序列如SEQ ID No.15所示的丙酮酸脱羧酶W392M的基因，并将其与质粒pET-28a(+)相连，获得重组质粒pET-28a(+)-pdc，此步骤由南京金斯瑞生物科技有限公司完成；其中，丙酮酸脱羧酶W392M是通过在氨基酸序列如SEQ ID No.16所示的丙酮酸脱羧酶的基础上，将其第392位由色氨酸突变为蛋氨酸得到的；
将获得的重组质粒pET-28a(+)-pdc导入大肠杆菌E.coli BL21(DE3)中；将转化后的大肠杆菌E.coli BL21(DE3)划线于含有50μg/mL卡那霉素的LB固体培养基上，于37℃的条件下培养20～24h；挑取阳性转化子接种于含有50μg/mL卡那霉素的LB液体培养基中，于37℃、200rpm的条件下培养12～14h，收集菌体，提取质粒，将质粒经限制性内切酶EcoR I和Xho I双酶切后进行电泳验证，并对质粒进行测序验证，验证正确，获得重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-pdc。
将重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-pdc划线于含有50μg/mL卡那霉素的LB固体培养基上，于37℃的条件下培养20～24h，获得单菌落；挑取单菌落接种于添加了50μg/mL卡那霉素的LB液体培养基中，于37℃、200rpm的条件下培养6～8h，获得种子液；将种子液以2～10％(v/v)的接种量转接到含50μg/mL卡那霉素的LB液体培养基中，于37℃、200rpm的条件下培养至OD在0.4～0.8之间后，在培养液中加入终浓度为0.1～1mmol/L的IPTG，于17～28℃的条件下继续进行诱导12～20h，获得发酵液；将发酵液进行离心，收集菌体，得到重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-pdc全细胞。
以苯甲醛(31.8g，0.3mol)和丙酮酸(17.6g，0.2mol)为底物，以磷酸盐缓冲液为溶剂，以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-pdc全细胞为催化剂，以硫胺素焦磷酸为辅酶，以MgCl2和二甲基亚砜为助溶剂，获得300mL的反应体系；将反应体系于温度为25℃、pH值为6.5、转速为200rpm的条件下进行反应20h，获得含有(R)-1-苯基2-羰基-3-丙醇的反应液；反应体系中，重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-pdc全细胞的浓度为50g/L，硫胺素焦磷酸的浓度为2.5mmol/L，MgCl2的浓度为5mmol/L，二甲基亚砜的浓度为5～20％(v/v)。检测反应液中(R)-1-苯基2-羰基-3-丙醇的产率和光学纯度，结果发现：产率为31.4～86.7％，光学纯度为95.7～99.4％((R)-1-苯基2-羰基-3-丙醇的纯化方法参考文献：李继珩.L-苯基乙酰基甲醇分离纯化及性质研究[J].化学世界,2002年,第3期:127-129.)。
下述实施例中涉及的麻黄碱的纯化方法如下：
将反应液离心收集上清，将上清用浓度为10mol/L的NaOH溶液调节pH为8，用旋转蒸发仪将上清浓缩至体积为1/2，再用浓度为10mol/L的HCl溶液调节pH为7，0～8℃下放置12～14h，过滤收集固体，将所得固体融入浓度为1mol/L的NaOH溶液中，再用浓度为10mol/L的HCl溶液调节pH为7，加入乙醇进行结晶，收集结晶，洗涤后进行真空干燥，得到麻黄碱晶体。
实施例1：可表达亚胺还原酶的基因工程菌的构建
化学合成编码氨基酸序列分别如SEQ ID No.1～SEQ ID No.14所示的亚胺还原酶的基因，并将其与质粒pET-28a(+)相连，获得重组质粒pET-28a(+)-ired1～pET-28a(+)-ired14，此步骤由南京金斯瑞生物科技有限公司完成；
将获得的重组质粒pET-28a(+)-ired1～pET-28a(+)-ired14分别导入大肠杆菌E.coli BL21(DE3)中；将转化后的大肠杆菌E.coli BL21(DE3)划线于含有50μg/mL卡那霉素的LB固体培养基上，于37℃的条件下培养20～24h；挑取阳性转化子接种于含有50μg/mL卡那霉素的LB液体培养基中，于37℃、200rpm的条件下培养12～14h，收集菌体，提取质粒，将质粒经限制性内切酶EcoR I和Xho I双酶切后进行电泳验证，并对质粒进行测序验证，验证正确，获得重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired1～重组大肠杆菌E.coliBL21(DE3)/pET-28a(+)-ired14。
将重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired1～重组大肠杆菌E.coliBL21(DE3)/pET-28a(+)-ired14分别划线于含有50μg/mL卡那霉素的LB固体培养基上，于37℃的条件下培养20～24h，获得单菌落；挑取单菌落接种于添加了50μg/mL卡那霉素的LB液体培养基中，于37℃、200rpm的条件下培养6～8h，获得种子液；将种子液以2～10％(v/v)的接种量转接到含50μg/mL卡那霉素的LB液体培养基中，于37℃、200rpm的条件下培养至OD在0.4～0.8之间后，在培养液中加入终浓度为0.1～1mmol/L的IPTG，于17～28℃的条件下继续进行诱导12～20h，获得发酵液；将发酵液进行离心，收集菌体，得到重组大肠杆菌E.coliBL21(DE3)/pET-28a(+)-ired1～重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired14全细胞。
实施例2：麻黄碱的制备
以含有空质粒pET-28a(+)的大肠杆菌E.coli BL21(DE3)为空白对照，以(R)-1-苯基-2-羰基3-丙醇(30g，0.2mol)、甲胺(62g，2mol)和葡萄糖(36g，0.2mol)为底物，以磷酸盐缓冲液为溶剂，分别以实施例1获得的重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired1～重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired14全细胞和葡萄糖脱氢酶共同作为催化剂，以NADP+为辅酶，以二甲基亚砜为助溶剂，获得300mL的反应体系；将反应体系于温度为25℃、pH值为8.5、转速为200rpm的条件下进行反应18h，获得含有麻黄碱的反应液；反应体系中，重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired全细胞的浓度为34g/L，NADP+的浓度为0.4g/L，二甲基亚砜的浓度为10％(v/v)。检测反应液中麻黄碱的产率，结果发现：空白对照组获得的反应液中不能检测到麻黄碱；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired1～重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired10全细胞为催化剂获得的反应液中也不能检测到麻黄碱；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired11全细胞为催化剂时，产率为62.9～67.3％；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired12全细胞为催化剂时，产率为9.2～11.3％；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired13全细胞为催化剂时，产率为34.6～42.8％；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired14全细胞为催化剂时，产率为77.9～86.5％。
将以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired11～重组大肠杆菌E.coliBL21(DE3)/pET-28a(+)-ired14全细胞为催化剂时获得的反应液中的麻黄碱进行纯化，得到麻黄碱晶体。检测麻黄碱晶体的光学纯度，结果发现：光学纯度为99.1～99.6％。
实施例3：麻黄碱的制备
以苯甲醛(31.8g，0.3mol)、丙酮酸(17.6g，0.2mol)、甲胺(31g，1mol)和葡萄糖(45g，0.25mol)为底物，以磷酸盐缓冲液为溶剂，分别以实施例1获得的重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired11～重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired14全细胞、可表达丙酮酸脱羧酶的基因工程菌和葡萄糖脱氢酶共同作为催化剂，以焦磷酸硫胺素和NADP+为辅酶，以二甲基亚砜为助溶剂，获得300mL的反应体系；将反应体系于温度为25℃、pH值为7.5、转速为200rpm的条件下进行反应26h，获得含有麻黄碱的反应液；反应体系中，可表达丙酮酸脱羧酶的基因工程菌的浓度为50g/L，重组大肠杆菌E.coliBL21(DE3)/pET-28a(+)-ired全细胞的浓度为42g/L，焦磷酸硫胺素的浓度为3.2mmol/L，NADP+的浓度为0.45g/L，二甲基亚砜的浓度为10％(v/v)。检测反应液中麻黄碱的产率，结果发现：以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired11全细胞为催化剂时，产率为42.6～59.7％；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired12全细胞为催化剂时，产率为3.1～6.2％；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired13全细胞为催化剂时，产率为19.8～22.6％；以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired14全细胞为催化剂时，产率为48.5～62.7％。
将以重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired11～重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-ired14全细胞为催化剂时获得的反应液中的麻黄碱进行纯化，得到麻黄碱晶体。检测麻黄碱晶体的光学纯度，结果发现：光学纯度为81.4～92.1％。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
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<110> 江南大学
<120> 一种利用亚胺还原酶生产麻黄碱的方法
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<220>
<221> misc_feature
<222> (1)..(1)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (85)..(85)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (126)..(126)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (187)..(187)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (279)..(279)
<223> Xaa can be any naturally occurring amino acid
<400> 9
Xaa Ser Leu Ser Asp Glu Ser Phe Glu Phe Asp Val Ser Val Ile Gly
1 5 10 15
Leu Gly Ala Xaa Gly Thr Ile Met Ala Gln Val Leu Leu Lys Gln Gly
20 25 30
Lys Arg Val Ala Ile Trp Asn Arg Ser Pro Gly Lys Ala Ala Ala Leu
35 40 45
Val Ala Ala Gly Ala His Leu Cys Glu Ser Val Lys Ala Ala Leu Ser
50 55 60
Ala Ser Pro Ala Thr Ile Phe Val Leu Leu Asp Asn His Ala Thr His
65 70 75 80
Glu Val Leu Gly Xaa Pro Gly Val Ala Arg Ala Leu Ala His Arg Thr
85 90 95
Ile Val Asp Tyr Thr Thr Asn Ala Gln Asp Glu Gly Leu Ala Leu Gln
100 105 110
Gly Leu Val Asn Gln Ala Gly Gly His Tyr Val Lys Gly Xaa Ile Val
115 120 125
Ala Tyr Pro Arg Asn Val Gly His Arg Glu Ser His Ser Ile His Thr
130 135 140
Gly Asp Arg Glu Ala Phe Glu Gln His Arg Ala Leu Leu Glu Gly Leu
145 150 155 160
Ala Gly His Thr Val Phe Leu Pro Trp Asp Glu Ala Leu Ala Phe Ala
165 170 175
Thr Val Leu His Ala His Ala Phe Ala Ala Xaa Val Thr Phe Phe Glu
180 185 190
Ala Val Gly Ala Gly Asp Arg Phe Gly Leu Pro Val Ser Lys Thr Ala
195 200 205
Arg Leu Leu Leu Glu Thr Ser Arg Phe Phe Val Ala Asp Ala Leu Glu
210 215 220
Glu Ala Val Arg Arg Leu Glu Thr Gln Asp Phe Lys Gly Asp Gln Ala
225 230 235 240
Arg Leu Asp Val His Ala Asp Ala Phe Ala His Ile Ala Gln Ser Leu
245 250 255
His Ala Gln Gly Val Trp Thr Pro Val Phe Asp Ala Val Cys Gln Val
260 265 270
Val Gln Arg Ala Ala Ala Xaa Gly Tyr Gly Asp Gln Asp Ile Ala Ala
275 280 285
Thr Thr Lys Ser Phe Ala Arg Glu Gln Glu Glu Gly His His His His
290 295 300
His His
305
<210> 10
<211> 290
<212> PRT
<213> 人工序列
<400> 10
Met Thr Asp Gln Asn Leu Pro Val Thr Val Ala Gly Leu Gly Pro Met
1 5 10 15
Gly Ser Ala Leu Ala Ala Ala Leu Leu Asp Arg Gly His Asp Val Thr
20 25 30
Val Trp Asn Arg Ser Pro Gly Lys Ala Ala Pro Leu Val Ala Lys Gly
35 40 45
Ala Arg Gln Ala Asp Asp Ile Val Asp Ala Val Ser Ala Ser Arg Leu
50 55 60
Leu Val Val Cys Leu Ala Asp Tyr Asp Ala Leu Tyr Ser Ala Leu Gly
65 70 75 80
Pro Ala Arg Glu Ala Leu Arg Gly Arg Val Val Val Asn Leu Asn Ser
85 90 95
Gly Thr Pro Lys Glu Ala Asn Glu Ala Leu Arg Trp Ala Glu Arg His
100 105 110
Gly Thr Gly Tyr Leu Asp Gly Ala Ile Met Val Pro Pro Ala Met Val
115 120 125
Gly His Pro Gly Ser Val Phe Leu Tyr Ser Gly Ser Ala Glu Val Phe
130 135 140
Glu Glu Tyr Lys Glu Thr Leu Ala Gly Leu Gly Asp Pro Val His Leu
145 150 155 160
Gly Thr Glu Ala Gly Leu Ala Val Leu Tyr Asn Thr Ala Leu Leu Ser
165 170 175
Met Met Tyr Ser Ser Met Asn Gly Phe Leu His Ala Ala Ala Leu Val
180 185 190
Gly Ser Ala Gly Val Pro Ala Ala Glu Phe Thr Lys Leu Ala Val Asp
195 200 205
Trp Phe Leu Pro Ala Val Ile Gly Gln Ile Ile Lys Ala Glu Ala Pro
210 215 220
Thr Ile Asp Glu Gly Val Tyr Pro Gly Asp Ala Gly Ser Leu Glu Met
225 230 235 240
Asn Val Thr Thr Leu Lys His Ile Ile Gly Thr Ser Gln Glu Gln Gly
245 250 255
Val Asp Thr Glu Ile Pro Val Arg Asn Lys Glu Leu Leu Asp Arg Ala
260 265 270
Val Ala Ala Gly Phe Gly Glu Ser Ser Tyr Tyr Ser Val Ile Glu Leu
275 280 285
Trp Arg
290
<210> 11
<211> 294
<212> PRT
<213> 人工序列
<400> 11
Met Lys Arg Ser Ile Thr Val Leu Gly Thr Gly Arg Met Gly Ser Ala
1 5 10 15
Leu Ala Arg Ala Leu Leu His Ala Gly His Arg Thr Thr Val Trp Asn
20 25 30
Arg Thr Ile Gln Lys Ala Glu Pro Leu Ala Ala Leu Gly Ala Thr Val
35 40 45
Ala Pro Ser Val Leu Glu Ala Val Asn Ala Ala Glu Ile Ile Ile Val
50 55 60
Asn Val Ser Asp Tyr Gln Ala Thr Ala Ala Ile Met Arg Asn Asp Ala
65 70 75 80
Ile Ala Ser Ala Val Arg Gly Lys Leu Ile Val Glu Leu Thr Ser Gly
85 90 95
Thr Pro His Gly Ala Arg Glu Ala Ala Glu Phe Trp Ala Glu His Gly
100 105 110
Ala Ser Tyr Leu Asp Gly Ala Ile Met Ala Thr Pro Asp Phe Ile Gly
115 120 125
Thr Asp Ala Gly Thr Ile Leu Val Ser Gly Ser Ser Gln Ala Phe Asp
130 135 140
Ala Asn Glu Asp Met Phe Arg Ala Leu Gly Gly Asn Val Gln His Ile
145 150 155 160
Gly Glu Glu Ser Gly Arg Ala Asn Ala Leu Asp Ser Ala Leu Leu Ala
165 170 175
Leu Met Trp Gly Ala Leu Phe Gly Thr Leu His Ala Ile Ala Val Cys
180 185 190
Gln Ala Glu Glu Ile Asp Leu Gly Glu Leu Ala Gln Gln Trp Asn Ala
195 200 205
Thr Ala Pro Val Val Glu Gly Leu Val Ala Asp Leu Ile Lys Arg Thr
210 215 220
Asn Ala Gly Arg Phe Ala Ser Asp Asp Glu Thr Leu Ser Ser Ile Ser
225 230 235 240
Ala His Tyr Gly Ala Phe Gln His Leu Leu Glu Leu Met Glu Ala Arg
245 250 255
Glu Ile Asp Arg Ser Val Val Leu Gly Tyr Asp Ala Ile Phe Gln Arg
260 265 270
Ala Ile Ala Ala Gly Gln Leu His Glu Asp Phe Ala Ala Leu Ser Gln
275 280 285
Phe Leu Gly Lys Ser Ala
290
<210> 12
<211> 293
<212> PRT
<213> 人工序列
<400> 12
Met Lys Leu Ser Ile Ser Val Val Gly Thr Gly Arg Met Gly Ser Ala
1 5 10 15
Leu Ala Arg Ala Leu Leu Arg Ser Gly Tyr Arg Thr Thr Val Trp Asn
20 25 30
Arg Thr Lys Gln Lys Ala Glu Pro Leu Ala Ala Leu Gly Ala Thr Val
35 40 45
Ala Gly Ser Val Leu Glu Ala Leu Asn Ala Ala Glu Ile Ile Ile Val
50 55 60
Asn Val Ser Asp Tyr Gln Ala Ser Ala Val Leu Arg Asp Asp Ala Val
65 70 75 80
Ala Ser Ala Ile Cys Gly Lys Leu Ile Val Glu Leu Thr Ser Gly Thr
85 90 95
Pro His Gly Ala Arg Glu Ala Ala Glu Trp Phe Ala Ala His Gly Ala
100 105 110
Ser Tyr Leu Asp Gly Ala Ile Met Ala Thr Pro Asp Phe Ile Gly Thr
115 120 125
Asp Ala Gly Thr Ile Leu Ile Ser Gly Pro Arg Gln Ala Phe Asp Ala
130 135 140
Asn Glu Glu Met Phe Arg Ala Leu Gly Gly Asn Val Gln His Val Gly
145 150 155 160
Glu Glu Pro Gly Arg Ala Asn Ala Leu Asp Ser Ala Leu Leu Ala Leu
165 170 175
Met Trp Gly Ala Leu Phe Gly Thr Leu His Ala Ile Ala Val Cys Gln
180 185 190
Ala Glu Glu Ile Asp Leu Gly Glu Leu Gly Arg Gln Trp Thr Ala Ile
195 200 205
Ala Pro Val Ile Asp Gly Leu Val Ala Asp Leu Ile Lys Arg Thr Asp
210 215 220
Thr Gly Arg Phe Ala Ser Asp Asn Glu Thr Leu Ser Ser Ile Ser Pro
225 230 235 240
His Tyr Gly Ala Phe Gln His Leu Leu Glu Leu Met Glu Ala Arg Lys
245 250 255
Ile Asp Arg Ser Val Val Asp Gly Tyr Asp Ala Ile Phe Gln Arg Ala
260 265 270
Ile Ala Ala Gly His Leu His Asp Asp Phe Ala Ala Leu Ser Gln Phe
275 280 285
Leu Gly Lys Ser Arg
290
<210> 13
<211> 289
<212> PRT
<213> 人工序列
<400> 13
Met Ser Lys His Ile Gly Ile Phe Gly Leu Gly Ala Met Gly Thr Ala
1 5 10 15
Leu Ala Ala Lys Tyr Leu Glu His Gly Tyr Lys Thr Ser Val Trp Asn
20 25 30
Arg Thr Thr Ala Lys Ala Ile Pro Leu Val Glu Gln Gly Ala Lys Leu
35 40 45
Ala Ser Thr Ile Ser Glu Gly Val Asn Ala Asn Asp Leu Ile Ile Ile
50 55 60
Cys Leu Leu Asn Asn Gln Val Val Glu Asp Ala Leu Arg Asp Ala Leu
65 70 75 80
Gln Thr Leu Pro Ser Lys Thr Ile Val Asn Leu Thr Asn Gly Thr Pro
85 90 95
Asn Gln Ala Arg Lys Leu Ala Asp Phe Val Thr Ser His Gly Ala Arg
100 105 110
Tyr Ile His Gly Gly Ile Met Ala Val Pro Thr Met Ile Gly Ser Pro
115 120 125
His Ala Val Leu Leu Tyr Ser Gly Glu Ser Leu Glu Leu Phe Gln Ser
130 135 140
Ile Glu Ser His Leu Ser Leu Leu Gly Met Ser Lys Tyr Leu Gly Thr
145 150 155 160
Asp Ala Gly Ser Ala Ser Leu His Asp Leu Ala Leu Leu Ser Gly Met
165 170 175
Tyr Gly Leu Phe Ser Gly Phe Leu His Ala Val Ala Leu Ile Lys Ser
180 185 190
Gly Gln Asp Thr Ser Thr Thr Ala Thr Gly Leu Leu Pro Leu Leu Thr
195 200 205
Pro Trp Leu Ser Ala Met Thr Gly Tyr Leu Ser Ser Ile Ala Lys Gln
210 215 220
Ile Asp Asp Gly Asp Tyr Ala Thr Gln Gly Ser Asn Leu Gly Met Gln
225 230 235 240
Leu Ala Gly Val Glu Asn Ile Ile Arg Ala Gly Glu Glu Gln Arg Val
245 250 255
Ser Ser Gln Met Ile Leu Pro Ile Lys Ala Leu Ile Glu Gln Ala Val
260 265 270
Gly Glu Gly His Gly Gly Glu Asp Leu Ser Ala Leu Ile Glu Tyr Phe
275 280 285
Lys
<210> 14
<211> 297
<212> PRT
<213> 人工序列
<400> 14
Met Ser Asn Thr Lys Ala Ala Gln Ala Pro Val Ser Val Ile Gly Leu
1 5 10 15
Gly Leu Met Gly Gln Ala Leu Ala Ala Ala Phe Leu Lys Ala Gly His
20 25 30
Pro Thr Thr Val Trp Asn Arg Thr Ala Ala Lys Ala Asp Gln Leu Val
35 40 45
Gly Glu Gly Ala Ala Leu Ala Gly Ser Thr Ala Asp Ala Ile Ala Ala
50 55 60
Ser Pro Leu Val Val Val Cys Val Thr Asp Tyr Thr Ala Val Arg Glu
65 70 75 80
Leu Leu Asp Pro Leu Ala Gly Ala Leu Lys Gly Lys Val Leu Val Asn
85 90 95
Leu Thr Thr Gly Thr Ser Thr Gln Ala Arg Glu Thr Ala Glu Trp Ala
100 105 110
Ala Asp Lys Glu Ile Thr Tyr Leu Asp Gly Ala Ile Met Ala Ile Pro
115 120 125
Pro Asp Ile Ala Thr Asp Ala Ala Val Leu Leu Tyr Ser Gly Pro Lys
130 135 140
Ala Ala Phe Asp Glu His Glu Ala Thr Leu Arg Ala Leu Gly Ala Ala
145 150 155 160
Gly Thr Thr Tyr Leu Asp Thr Asp His Gly Leu Ser Ala Leu Tyr Asp
165 170 175
Met Ser Leu Leu Gly Ile Met Trp Gly Ile Leu Asn Gly Phe Leu His
180 185 190
Gly Ala Ala Leu Leu Gly Thr Ala Glu Val Lys Ala Thr Thr Phe Ala
195 200 205
Pro Leu Ala Asn Thr Met Ile Asn Val Val Thr Glu Tyr Val Thr Ala
210 215 220
Tyr Ala Pro Gln Ile Asp Glu Gly Lys Tyr Pro Ala Gly Asp Ala Thr
225 230 235 240
Met Thr Val His Gln Asp Ala Leu Glu His Leu Ala Glu Glu Ser Glu
245 250 255
Thr Leu Gly Ile Asn Ala Glu Met Pro Arg Phe Phe Lys Ala Leu Val
260 265 270
Asp Arg Ser Val Ala Ala Gly His Ala Glu Ser Gly Tyr Ala Ala Leu
275 280 285
Ile Glu Gln Phe Arg Lys Pro Ala Val
290 295
<210> 15
<211> 568
<212> PRT
<213> 人工序列
<400> 15
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Ala Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Met Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565
<210> 16
<211> 568
<212> PRT
<213> 人工序列
<400> 16
Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile
1 5 10 15
Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu
20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys
35 40 45
Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60
Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala
65 70 75 80
Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu
85 90 95
Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu
100 105 110
His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala
115 120 125
Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala
130 135 140
Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys
145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys Ala
165 170 175
Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190
Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe Ile Ala Asn
195 200 205
Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly
210 215 220
Ala Glu Glu Ala Ala Val Lys Phe Ala Asp Ala Leu Gly Gly Ala Val
225 230 235 240
Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His
245 250 255
Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys
260 265 270
Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu
290 295 300
Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320
Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser
325 330 335
Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu
340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala
355 360 365
Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val
370 375 380
Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu
385 390 395 400
Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly
405 410 415
Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430
Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln
435 440 445
Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu
450 455 460
Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly Pro
465 470 475 480
Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495
Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys Ala
500 505 510
Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525
Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys
530 535 540
Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560
Arg Lys Pro Val Asn Lys Leu Leu
565

Claims (10)

1.一种生产麻黄碱的方法，其特征在于，所述方法为以(R)-1-苯基-2-羰基-3-丙醇与甲胺为反应原料，将(R)-1-苯基-2-羰基-3-丙醇与甲胺在亚胺还原酶或表达亚胺还原酶的基因工程菌的作用下进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱；
或者，所述方法为以苯甲醛、丙酮酸与甲胺为反应原料，将苯甲醛、丙酮酸与甲胺在亚胺还原酶或表达亚胺还原酶的基因工程菌，和，丙酮酸脱羧酶或表达丙酮酸脱羧酶的基因工程菌的共同作用下进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱。
2.如权利要求1所述的一种生产麻黄碱的方法，其特征在于，所述亚胺还原酶为氨基酸序列如SEQ ID No.11、SEQ ID No.12、SEQ ID No.13或SEQ ID No.14所示的亚胺还原酶中的一种或多种。
3.如权利要求1或2所述的一种生产麻黄碱的方法，其特征在于，所述丙酮酸脱羧酶为氨基酸序列如SEQ ID No.15或SEQ ID No.16所示的亚胺还原酶中的一种或多种。
4.如权利要求1-3任一所述的一种生产麻黄碱的方法，其特征在于，所述方法为以(R)-1-苯基-2-羰基-3-丙醇与甲胺为反应原料，先将(R)-1-苯基-2-羰基-3-丙醇与甲胺加入缓冲液中，得到反应体系，然后在反应体系中加入亚胺还原酶或表达亚胺还原酶的基因工程菌进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱；
或者，所述方法为以苯甲醛、丙酮酸与甲胺为反应原料，先将苯甲醛、丙酮酸与甲胺加入缓冲液中，得到反应体系，然后在反应体系中加入亚胺还原酶或表达亚胺还原酶的基因工程菌，和，丙酮酸脱羧酶或表达丙酮酸脱羧酶的基因工程菌进行反应，得到(1R，2S)-2-甲氨基-苯丙烷-1-醇，即麻黄碱。
5.如权利要求4所述的一种生产麻黄碱的方法，其特征在于，当反应原料为(R)-1-苯基-2-羰基-3-丙醇与甲胺时，所述反应体系中还含有葡萄糖脱氢酶、葡萄糖、第一辅酶以及二甲基亚砜，或者，所述反应体系中还含有表达葡萄糖脱氢酶的基因工程菌、葡萄糖、第一辅酶以及二甲基亚砜；
当反应原料为苯甲醛、丙酮酸与甲胺时，所述反应体系中还含有葡萄糖脱氢酶、葡萄糖、第一辅酶、第二辅酶以及二甲基亚砜，或者，所述反应体系中还含有表达葡萄糖脱氢酶的基因工程菌、葡萄糖、第一辅酶、第二辅酶以及二甲基亚砜。
6.如权利要求5所述的一种生产麻黄碱的方法，其特征在于，所述第一辅酶为NADPH和/或NADP+。
7.如权利要求5或6所述的一种生产麻黄碱的方法，其特征在于，所述第二辅酶为焦磷酸硫胺素。
8.如权利要求4-7任一所述的一种生产麻黄碱的方法，其特征在于，所述反应体系中，亚胺还原酶的浓度为1.2～30U/mL。
9.如权利要求4-8任一所述的一种生产麻黄碱的方法，其特征在于，所述反应体系中，可表达亚胺还原酶的基因工程菌的浓度为16～50g/L。
10.亚胺还原酶或表达亚胺还原酶的基因工程菌或权利要求1-9任一所述的一种生产麻黄碱的方法在生产麻黄碱中的应用。
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CN112779300A (zh) * 2020-12-15 2021-05-11 上海合全药物研发有限公司 生物催化制备(s)-3-丁炔-2-丙炔胺的方法
CN112795603A (zh) * 2020-12-14 2021-05-14 山东金城医药化工有限公司 一种制备(s)-2-(3-吡啶)-吡咯烷的方法
CN112852771A (zh) * 2020-04-17 2021-05-28 中国科学院天津工业生物技术研究所 酶催化合成手性氨基醇化合物
CN114317472A (zh) * 2021-12-31 2022-04-12 长兴制药股份有限公司 一种高立体选择性亚胺还原酶及其制备方法和应用
WO2022268006A1 (zh) * 2021-06-20 2022-12-29 陈泽聪 亚胺还原酶突变体、亚胺还原酶和葡萄糖脱氢酶共表达酶及其应用

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