LAUSR

Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high‐concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on “open to closed” conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid (BFA) for producing l‐phenylglycine (l‐Phg), was directly identified from 4695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4253.8 U mg−1. Through reaction process optimization, a high‐concentration substrate (0.7 m) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow‐feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 m BFA could be aminated to l‐Phg with conversion of >99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large‐scale application of l‐Phg.