Genome Data Mining Of Leucine Dehydrogenase And Its Catalytic Performance In Reductive Amination Of Trimethylpyruvic Acid To L-tert-Leucine

Enantiometrically pure L-tert-eucine is one of the important chiral unnatural amino acids. L-tert-leucine is a key intermediate for the synthesis of various pharmaceuticals such as anti-tumor agents or HIV protease inhibitors and is widely used in feed, food, cosmetic, agriculture, chemistry and so on. Reductive amination of prochiral ketonic acids with amino acid dehydrogenases (LeuDHs) is an effective approach to producing unnatural amino acids since it can be carried out under mild reaction conditions, affording enantiopure products with a high yield.By using genome data mining methodology, we obtained13LeuDHs showing activities towards trimethylpyruvic acid (TMP). Finally, the best LeuDH (EsLeuDH) from Exiguobacterium sibiricum was overexpressed in E. coli BL21, purified to homogeneity and its catalytic properties were studied. The maximum activity is observed at pH9.5and the optimum temperature is found to be45℃. The enzyme is very stable, and the half-life at30℃is433.2h, even as long as3h at60℃. Most importantly, EsLeuDH exhibits good activity at temperatures as low as0℃. The Km values of EsLeuDH for its natural substrate L-leucine and unnatural substrate TMP are0.8mM and16.8mM respectively, and the corresponding Vmax is20.0μmol·min-1·mg-1and200.0μmol·min-1·mg-1respectively.This enzyme shows good activity towards a broad range of aliphatic ketonic acids, and even some aryl ketonic acids. Among them, TMP was efficiently converted to L-tert-leucine, an important pharmaceutical intermediate, in1L reaction volume. As much as78.08g·L-1(0.6M) of TMP was reductively aminated within5.5h using whole cells coulpled with glucose dehydrogenase for NADH regeneration, resulting in a yield of80.0%with>99%ee, and a total turnover number (TTN) of55,200. The operational stability is also good and the residual activity is46%after24h. These results indicate the potential of EsLeuDH for industrial production of valuable L-teueucine.