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Leucine dehydrogenase (LeuDH) has emerged as the most promising biocatalyst for L-tert-leucine (L-Tle) production via asymmetric reduction in trimethylpyruvate (TMP). In this study, a new LeuDH named PbLeuDH from marine Pseudomonas balearica was heterologously over-expressed in Escherichia coli, followed by purification and characterization. PbLeuDH possessed a broad substrate scope, displaying activities toward numerous L-amino acids and α-keto acids. Notably, compared with those reported LeuDHs, PbLeuDH exhibited excellent catalytic efficiency for TMP with a Km value of 4.92 mM and a kcat/Km value of 24.49 s−1 mM−1. Subsequently, L-Tle efficient production was implemented from TMP by whole-cell biocatalysis using recombinant E. coli as a catalyst, which co-expressed PbLeuDH and glucose dehydrogenase (GDH). Ultimately, using a fed-batch feeding strategy, 273 mM (35.8 g L−1) L-Tle was achieved with a 96.1% yield and 2.39 g L−1 h−1 productivity. In summary, our research provides a competitive biocatalyst for L-Tle green biosynthesis and lays a solid foundation for the realization of large-scale L-Tle industrial production. [ABSTRACT FROM AUTHOR]

Leucine dehydrogenase (LeuDH) has emerged as the most promising biocatalyst for L-tert-leucine (L-Tle) production via asymmetric reduction in trimethylpyruvate (TMP). In this study, a new LeuDH named PbLeuDH from marine Pseudomonas balearica was heterologously over-expressed in Escherichia coli, followed by purification and characterization. PbLeuDH possessed a broad substrate scope, displaying activities toward numerous L-amino acids and α-keto acids. Notably, compared with those reported LeuDHs, PbLeuDH exhibited excellent catalytic efficiency for TMP with a Km value of 4.92 mM and a kcat/Km value of 24.49 s−1 mM−1. Subsequently, L-Tle efficient production was implemented from TMP by whole-cell biocatalysis using recombinant E. coli as a catalyst, which co-expressed PbLeuDH and glucose dehydrogenase (GDH). Ultimately, using a fed-batch feeding strategy, 273 mM (35.8 g L−1) L-Tle was achieved with a 96.1% yield and 2.39 g L−1 h−1 productivity. In summary, our research provides a competitive biocatalyst for L-Tle green biosynthesis and lays a solid foundation for the realization of large-scale L-Tle industrial production.

Background: L-tert-Leucine has been widely used in pharmaceutical, chemical, and other industries as a vital chiral intermediate. Compared with chemical methods, enzymatic methods to produce L-tert-leucine have unparalleled advantages. Previously, we found a novel leucine dehydrogenase from the halophilic thermophile Laceyella sacchari (LsLeuDH) that showed good thermostability and great potential for the synthesis of L-tertleucine in the preliminary study. Hence, we manage to use the LsLeuDH coupling with a formate dehydrogenase from Candida boidinii (CbFDH) in the biosynthesis of L-tert-leucine through reductive amination in the present study. Result: The double-plasmid recombinant strain exhibited higher conversion than the single-plasmid recombinant strain when resting cells cultivated in shake flask for 22 h were used. Under the optimized conditions, the double-plasmid recombinant E. coli BL21 (pETDute-FDH-LDH, pACYCDute-FDH) transformed 1 mol·L-1 trimethylpyruvate (TMP) completely into L-tert-leucine with greater than 99.9% ee within 8 h. Conclusions: The LsLeuDH showed great ability to biosynthesize L-tert-leucine. In addition, it provided a new option for the biosynthesis of L-tert-leucine. [ABSTRACT FROM AUTHOR]

l- tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare l- tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the l- tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of l- tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies ( k/ K) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of l- tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme. [ABSTRACT FROM AUTHOR]