Direct reductive amination of ketones by reductive aminases (RedAms) is a promising method for the synthesis of primary, secondary and tertiary amines. In this work, five naturally occurring RedAms possessing reductive amination activity were identified using a structure-guided genome mining (SGGM) approach, which was based on highly conserved substrate binding and catalysis motif alignment. This RedAm toolbox facilitated the reductive amination of cyclohexanone and 4-fluoropropiophenone with various amine nucleophiles. In a preparative biotransformation (100 mg) with low stoichiometric ammonia donor and low catalyst loading, and three types of N-alkylcyclohexylamine were efficiently synthesized with a space-time yield up to 64.2 g L-1 d-1, demonstrating the potential of the RedAm enzymes as a biocatalyst toolbox for the synthesis of primary and secondary amines. Additionally, through protein engineering, the W205F variant of KfRedAm from Kribbella flavida was obtained with a 2-fold increased catalytic efficiency (kcat/Km), and the general applicability of the variant was verified, providing useful guidance for further evolution of RedAms within this family.

中文翻译：

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用还原胺酶将酮与胺直接还原胺化

通过还原氨基酶 (RedAms) 对酮进行直接还原胺化是合成伯胺、仲胺和叔胺的一种很有前景的方法。在这项工作中，使用基于高度保守的底物结合和催化基序比对的结构引导基因组挖掘 (SGGM) 方法鉴定了五种具有还原胺化活性的天然 RedAms。这个 RedAm 工具箱促进了环己酮和 4-氟苯丙酮与各种胺亲核试剂的还原胺化。在具有低化学计量氨供体和低催化剂负载的制备性生物转化（100 mg）中，高效合成了三种 N-烷基环己胺，时空产率高达 64.2 g L-1 d-1，展示了 RedAm 酶作为合成伯胺和仲胺的生物催化剂工具箱的潜力。此外，通过蛋白质工程，获得了来自Kribbella flavida的KfRedAm的W205F变体，其催化效率（kcat/Km）提高了2倍，并验证了该变体的普遍适用性，为RedAms在此范围内的进一步进化提供了有用的指导。家庭。