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	Directed evolution of leucine dehydrogenase for improved efficiency of l-tert-leucine synthesis
	Zhu, Lin1,5; Wu, Zhe1; Jin, Jian-Ming2,3,4; Tang, Shuang-Yan1
刊名	Applied microbiology and biotechnology
	2016-07-01
卷号	100期号:13页码:5805-5813
关键词	Leucine dehydrogenase L-tert-leucine Trimethylpyruvate High-throughput screening Directed evolution
ISSN号	0175-7598
DOI	10.1007/s00253-016-7371-5
通讯作者	Tang, shuang-yan(tangsy@im.ac.cn)
英文摘要	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.
WOS关键词	FORMATE DEHYDROGENASE ; SUBSTRATE-SPECIFICITY ; REDUCTIVE AMINATION ; MICROBIAL ENZYMES ; BUILDING-BLOCKS ; AMINO-ACIDS ; STABILITY ; CHROMATOGRAPHY ; REGENERATION ; SITE
WOS研究方向	Biotechnology & Applied Microbiology
WOS类目	Biotechnology & Applied Microbiology
语种	英语
出版者	SPRINGER
WOS记录号	WOS:000378725700012
内容类型	期刊论文
URI标识	http://www.corc.org.cn/handle/1471x/2374699
专题	中国科学院大学
通讯作者	Tang, Shuang-Yan
作者单位	1.Chinese Acad Sci, Inst Microbiol, CAS Key Lab Microbial Physiol & Metab Engn, Beijing 100101, Peoples R China 2.Beijing Technol & Business Univ, Beijing Key Lab Plant Resources Res & Dev, Beijing 100048, Peoples R China 3.Yunnan Minzu Univ, Key Lab Ethn Med Resource Chem, State Ethn Affairs Commiss, Kunming 650500, Peoples R China 4.Yunnan Minzu Univ, Minist Educ, Kunming 650500, Peoples R China 5.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Zhu, Lin,Wu, Zhe,Jin, Jian-Ming,et al. Directed evolution of leucine dehydrogenase for improved efficiency of l-tert-leucine synthesis[J]. Applied microbiology and biotechnology,2016,100(13):5805-5813.
APA	Zhu, Lin,Wu, Zhe,Jin, Jian-Ming,&Tang, Shuang-Yan.(2016).Directed evolution of leucine dehydrogenase for improved efficiency of l-tert-leucine synthesis.Applied microbiology and biotechnology,100(13),5805-5813.
MLA	Zhu, Lin,et al."Directed evolution of leucine dehydrogenase for improved efficiency of l-tert-leucine synthesis".Applied microbiology and biotechnology 100.13(2016):5805-5813.

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