title: Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis.
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.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing, 100048, China.|Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming, 650500, China.
authors.forename: Jian-Ming
authors.initials: JM
authors.lastname: Jin
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. tangsy@im.ac.cn.
authors.forename: Shuang-Yan
authors.initials: SY
authors.lastname: Tang
mesh_terms: D001406:Bacillaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D007930:Leucine / Q000737:chemistry / Q000378:metabolism*; D050803:Leucine Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D050803:Leucine Dehydrogenase; D007930:Leucine
keywords: Directed evolution; High-throughput screening; L-tert-Leucine; Leucine dehydrogenase; Trimethylpyruvate