| 论文题目: | Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis |
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| 作者: | Zhu Lin, Wu Zhe, Jin Jian-Ming, and Tang Shuang-Yan*. |
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| 刊物名称: | Appl Microbiol Biotechnol |
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| 年份: | 2016 |
| 影响因子: | 3.848 |
| 论文下载: | 下载地址 |
| 摘要: | 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 alpha-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. |
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