Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, China.
Enzyme Microb Technol. 2020 Nov;141:109655. doi: 10.1016/j.enzmictec.2020.109655. Epub 2020 Sep 7.
The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare ω-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 ω-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 pro-sitagliptin ketone PTfpB (1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 g/L E. coli cells harboring M1 + M122H as biocatalyst. In the kilogram-scale 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 ≈32 g/(L·d).
为了对前手性酮进行不对称氨化以生产西他列汀中间体，需要构建一种对映体选择性的 ω- 氨基转移酶（ω-ATA）作为生物催化剂，因为自然界中很少有 ω-ATA 是对映体选择性的，而且大多数 ω-ATA 稳定性差，对大位阻的前手性酮的活性低。在此，从节杆菌（Arthrobacter cumminsii）ZJUT212（AcATA）中克隆了一个对映体选择性的 ω-ATA 基因，并在大肠杆菌中进行了表达。经过筛选，使用半理性蛋白质设计获得了最佳变体（M1+M122H 和 M1+T134G）。这些变体不仅表现出提高的活性和底物亲和力，而且在含有 20%二甲亚砜的水相中的稳定性也得到了增强。使用含有 M1+M122H 的 2 g/L 大肠杆菌细胞作为生物催化剂，在手性胺化反应中，50 g/L 的前手性西他列汀酮 PTfpB（1-[1-哌啶基]-4-[2,4,5-三氟苯基]-1,3-丁二酮）的转化率达到 92%，对映体过量值（ee 值）>99%。在公斤级实验中，当使用 50 kg 的 PTfpB 作为底物时，大约 40 kg 的（R）-APTfpB（ee 值>99%）在 30 h 内生成，时空产率达到约 32 g/（L·d）。
