Rational identification of a high catalytic efficiency leucine dehydrogenase and process development for efficient synthesis ofl‐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 4253.8 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.

A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex

“…The oxidation of formate to generate NADH is widely employed for NADH regeneration and is usually required to reduce the operational cost of biobased syntheses of chiral compounds in the drug synthesis industry. [10,11,12] FDH continuously generates NADH using inexpensive formate to support the main synthesis of dehydrogenase/reductase reactions. Using this approach, a lower amount of expensive NADH is used compared to the stoichiometric amount of NADH needed without a regenerating system.…”