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 gDCW/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).

中文翻译：

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创建稳健且 R 选择性的 ω-胺转氨酶，用于以千克规模不对称合成西格列汀中间体

创建 R 选择性 ω-胺转氨酶 (ω-ATA) 作为生物催化剂对于前手性酮的不对称胺化以产生西他列汀中间体至关重要，因为稀有的 ω-ATA 本质上是 R 选择性的，而且大多数稳定性差和对庞大的前手性酮的低活性。在这里，从康明斯节杆菌 ZJUT212 (AcATA) 中克隆出 R 选择性 ω-ATA 的基因并在大肠杆菌中表达。筛选后使用半合理蛋白质设计获得最佳变体（M1 + M122H 和 M1 + T134 G）。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% 二甲亚砜的水相中也提高了稳定性。50 g/L 前西格列汀酮 PTfpB (1-[1-哌啶基]-4-[2,4,5-三氟苯基]-1, 3-丁二酮）使用 2 gDCW/L 含有 M1 + M122H 作为生物催化剂的大肠杆菌细胞，达到 92%，具有极高的 ee>99%。在公斤级实验中，当使用 50 kg PTfpB 作为底物时，在 30 小时内产生了大约 40 kg (R)-APTfpB（ee > 99%）。此外，时空产率达到≈32 g/(L·d)。