Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale
Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale
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创建一种强大的 R 选择性 α-胺转氨酶，用于公斤级西格列汀中间体的不对称合成。
DOI：
10.1016/j.enzmictec.2020.109655
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发表时间：
2020-11-01
影响因子：
3.4
通讯作者：
Zheng, Yu-Guo
中科院分区：
文献类型：
--
作者：
Cheng, Feng;Chen, Xiu-Ling;Zheng, Yu-Guo
在前手性酮的不对称胺化合成西格列汀中间体过程中，R选择性(欧米伽胺转氨酶，omega-ATA)的生成是至关重要的，因为稀有的omega-ATA本质上是R选择性的，而且它们对体积较大的前手性酮的稳定性较差，活性较低。本研究从克氏节杆菌ZJUT212(AcATA)中克隆了一个R-选择性omega-ATA基因，并在大肠杆菌中进行了表达。经过筛选，采用半理性蛋白质设计获得了最佳突变体(M1+M122H和M1+T134G)。这些突变体不仅表现出更好的活性和底物亲和力，而且在含有20%二甲基亚砜的水相中也提高了稳定性。在50g/L催化剂上，不对称胺化反应的转化率可达92%，e.e值极高。以含m1+M122H的2g(D)(CW)/L大肠杆菌细胞为生物催化剂，转化率为99%。在公斤级实验中，约40公斤(R)-APTfpB(e.E.当以50 kg PTfpB为底物时，30h内可获得99%的转化率。此外，时空产率接近32g/(L.d)。
The creation of an R-selective (omega-amine transaminase (omega-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare omega-ATAs are R-selective in nature and most of them suffer from poor stability and low activity toward bulky prochiral ketones. Here, the gene of an R-selective omega-ATA was cloned from Arthrobacter cumminsii ZJUT212 (AcATA) and expressed in Escherichia coli. The best variants (M1 + M122H and M1 +T134 G) were obtained using a semi-rational protein design after screening. These variants not only exhibited improved activity and substrate affinity but also enhanced stability in aqueous phase containing 20 % dimethyl sulfoxide. The conversion of asymmetric amination on 50 g/L prositagliptin ketone PTfpB (1[1-piperidinyl]-442,4,5-trifluoropheny11-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 g(D)(CW)/L E. coli cells harboring Ml + M122H as biocatalyst. In the kilogramscale experiment, approximately 40 kg of (R)-APTfpB (e.e. >99 %) was produced within 30 h when 50 kg PTfpB was used as the substrate. Furthermore, the space-time yield reached approximate to 32 g/(L.d).