Rational identification of a high catalytic efficiency leucine dehydrogenase and process development for efficient synthesis of l-phenylglycine.

Abstract

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 4253.8Umg-1 . Through reaction process optimization, a high-concentration substrate (0.7m) was efficiently and completely converted within 90min 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 1.2m BFA could be aminated to l-Phg with conversion of >99% in 3h, demonstrating that this new combination of enzyme process development is promising for large-scale application of l-Phg.