Identification and Rational Engineering of a High Substrate‐Tolerant Leucine Dehydrogenase Effective for the Synthesis of L‐tert‐Leucine

Abstract The asymmetric synthesis of chiral amino acids by leucine dehydrogenases has great potential for industrialization; however, the inhibitory effect of high‐concentration substrates limits its large‐scale application. Herein, using structure‐guided genome mining based on sequence‐structure prediction of substrate tolerance and specificity, a novel leucine dehydrogenase ( La LeuDH) from Labrenzia aggregate was identified and characterized, which exhibited the highest substrate tolerance and excellent activity to trimethyl pyruvate, even at 1.5 M concentration. Moreover, based on coenzyme binding structural information and sequence alignment, directed evolution of La LeuDH was performed to increase affinity for NADH. The obtained variant D153 N/H191 N resulted in 25‐fold improved affinity for NADH, with 50‐fold enhanced catalytic efficiency (kcat/Km) of 40464.6 mM −1 s −1 . Finally, through a combination of the above two strategies, as high as 1.5 M substrate could be completely converted in 18 h without coenzyme addition, demonstrating that this engineered enzyme has promising prospects for industrialization.