AUTHOR=Luo Wei , Zhu Jing , Zhao Yuzheng , Zhang Huili , Yang Xue , Liu Yuantao , Rao Zhiming , Yu Xiaobin 

TITLE=Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.00186

DOI=10.3389/fbioe.2020.00186

ISSN=2296-4185

ABSTRACT=<p>Among many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from <italic>Exiguobacterium sibiricum</italic> (<italic>Esi</italic>LeuDH) was isolated by using genome mining strategy. <italic>Esi</italic>LeuDH was overexpressed in <italic>Escherichia coli</italic> BL21 (DE3), followed by purification and characterization. The high thermostability of the enzyme confers its half-life up to 14.7 h at 50°C. Furthermore, the substrate specificity shows a broad spectrum, including many L-amino acids and aliphatic α-keto acids, especially some aryl α-keto acids. This enzyme coupled with recombinant formate dehydrogenase (FDH) was used to catalyze trimethylpyruvic acid (TMP) through reductive amination to generate enantiopure L-<italic>tert</italic>-leucine (L-Tle). In order to overcome the substrate inhibition effect, a fed-batch feeding strategy was adopted to transform up to 0.8 M of TMP to L-Tle, with an average conversion rate of 81% and L-Tle concentration of 65.6 g⋅L<sup>–1</sup>. This study provides a highly efficient biocatalyst for the synthesis of L-Tle and lays the foundation for large-scale production and application of chiral non-natural amino acids.</p>