LAUSR

Transaminase (TA)‐catalyzed asymmetric amination is considered as a green chemistry approach to synthesize pharmaceutical analogs, but their ability to accept substrate for catalyzing sterically hindered ketones remains a challenge. Sitagliptin is an antihyperglycemic drug to treat type II diabetes. Herein, we exploited an efficient (R)‐selective TA to biosynthesize sitagliptin analog (R)‐3‐amino‐1‐morpholino‐4‐(2,4,5‐trifluorophenyl)butan‐1‐one. Starting from a previously constructed (R)‐ATA5, two rounds of directed evolution were performed through combining error‐prone PCR, site‐directed saturation and combinatorial mutagenesis. The resultant variant ATA5/F189H/S236T/M121H showed a 10.2‐fold higher activity and a 4‐fold improved half‐life at 45°C. Crucially, the variant was able to either catalyze the amination of 700 mM substrate with a conversion up to 93.1% and product e.e.> 99% in a cosolvent reaction system, or biotransform 200 mM substrate with a conversion of 97.6% and product e.e.> 99% in a cosolvent‐free system. Furthermore, the structural analysis gave insight into how the mutations affected enzymatic activity and thermostability. This study, which consists of constructing a robust (R)‐selective TA and the new synthesis route with the highest conversion ever reported, provides a reference for industrial manufacturing sitagliptin analog.