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

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

doi:10.3389/fbioe.2020.00186

Abstract

Among many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from Exiguobacterium sibiricum (EsiLeuDH) was isolated by using genome mining strategy. EsiLeuDH was overexpressed in Escherichia coli 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-tert-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–1. 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.

Highlights

With the continuous development of the synthesis and analytical technologies (Bommarius et al, 1995; Taylor et al, 1998; Miyoshi et al, 2014), a growing number of unnatural amino acids are produced and used to synthesize antitumor and physiologically active peptides
In order to obtain LeuDH discrepant in the catalytic properties, LeuDHs with identity of 20–100% to BspLeuDH in amino acid sequences were selected from the NCBI database
Ten LeuDH genes were expressed in E. coli cells, and four LeuDHs presenting high activities were analyzed regarding with their thermostability

Summary

Introduction

With the continuous development of the synthesis and analytical technologies (Bommarius et al, 1995; Taylor et al, 1998; Miyoshi et al, 2014), a growing number of unnatural amino acids are produced and used to synthesize antitumor and physiologically active peptides. Among these products with medicinal value, L-tert-Leucine (L-Tle) is an important non-natural amino acid, which is often used as a template to induce asymmetric synthesis and the product has high stereoselectivity (Lehr et al, 1996; Hong et al, 2010).

Methods

Results

Conclusion