1. Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis.
- Author
-
Zhu L, Wu Z, Jin JM, and Tang SY
- Subjects
- Bacillaceae chemistry, Bacillaceae genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Leucine chemistry, Leucine Dehydrogenase genetics, Leucine Dehydrogenase metabolism, Protein Engineering, Substrate Specificity, Bacillaceae enzymology, Bacterial Proteins chemistry, Directed Molecular Evolution methods, Leucine metabolism, Leucine Dehydrogenase chemistry
- Abstract
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 cat/K m) 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.
- Published
- 2016
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