Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production
Among many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from Exiguobacterium sibiricum (EsiLeuDH) was isolated by using genome mining strategy. EsiLeuDH was overexpressed in Escherichia coli BL21 (DE3), followed by purification and characterization. The high thermo...
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doaj-bc71b2a5cb384041b637d7a5221543ec2020-11-25T02:38:58ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852020-03-01810.3389/fbioe.2020.00186524743Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine ProductionWei Luo0Jing Zhu1Yuzheng Zhao2Huili Zhang3Xue Yang4Yuantao Liu5Zhiming Rao6Xiaobin Yu7The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaThe Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, ChinaCollege of Life Sciences, University of Shihezi, Shihezi, ChinaHulunbeier Northest Fufeng Biotechnologies Co., Ltd., Zhalantun, ChinaHulunbeier Northest Fufeng Biotechnologies Co., Ltd., Zhalantun, ChinaThe Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaThe Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, ChinaAmong many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from Exiguobacterium sibiricum (EsiLeuDH) was isolated by using genome mining strategy. EsiLeuDH was overexpressed in Escherichia coli BL21 (DE3), followed by purification and characterization. The high thermostability of the enzyme confers its half-life up to 14.7 h at 50°C. Furthermore, the substrate specificity shows a broad spectrum, including many L-amino acids and aliphatic α-keto acids, especially some aryl α-keto acids. This enzyme coupled with recombinant formate dehydrogenase (FDH) was used to catalyze trimethylpyruvic acid (TMP) through reductive amination to generate enantiopure L-tert-leucine (L-Tle). In order to overcome the substrate inhibition effect, a fed-batch feeding strategy was adopted to transform up to 0.8 M of TMP to L-Tle, with an average conversion rate of 81% and L-Tle concentration of 65.6 g⋅L–1. This study provides a highly efficient biocatalyst for the synthesis of L-Tle and lays the foundation for large-scale production and application of chiral non-natural amino acids.https://www.frontiersin.org/article/10.3389/fbioe.2020.00186/fullleucine dehydrogenasetrimethylpyruvic acidL-tert-leucinereductive aminationamino acid |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Wei Luo Jing Zhu Yuzheng Zhao Huili Zhang Xue Yang Yuantao Liu Zhiming Rao Xiaobin Yu |
spellingShingle |
Wei Luo Jing Zhu Yuzheng Zhao Huili Zhang Xue Yang Yuantao Liu Zhiming Rao Xiaobin Yu Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production Frontiers in Bioengineering and Biotechnology leucine dehydrogenase trimethylpyruvic acid L-tert-leucine reductive amination amino acid |
author_facet |
Wei Luo Jing Zhu Yuzheng Zhao Huili Zhang Xue Yang Yuantao Liu Zhiming Rao Xiaobin Yu |
author_sort |
Wei Luo |
title |
Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production |
title_short |
Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production |
title_full |
Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production |
title_fullStr |
Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production |
title_full_unstemmed |
Cloning and Expression of a Novel Leucine Dehydrogenase: Characterization and L-tert-Leucine Production |
title_sort |
cloning and expression of a novel leucine dehydrogenase: characterization and l-tert-leucine production |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Bioengineering and Biotechnology |
issn |
2296-4185 |
publishDate |
2020-03-01 |
description |
Among many genes encoding for amino acid dehydrogenase, a novel leucine dehydrogenase gene from Exiguobacterium sibiricum (EsiLeuDH) was isolated by using genome mining strategy. EsiLeuDH was overexpressed in Escherichia coli BL21 (DE3), followed by purification and characterization. The high thermostability of the enzyme confers its half-life up to 14.7 h at 50°C. Furthermore, the substrate specificity shows a broad spectrum, including many L-amino acids and aliphatic α-keto acids, especially some aryl α-keto acids. This enzyme coupled with recombinant formate dehydrogenase (FDH) was used to catalyze trimethylpyruvic acid (TMP) through reductive amination to generate enantiopure L-tert-leucine (L-Tle). In order to overcome the substrate inhibition effect, a fed-batch feeding strategy was adopted to transform up to 0.8 M of TMP to L-Tle, with an average conversion rate of 81% and L-Tle concentration of 65.6 g⋅L–1. This study provides a highly efficient biocatalyst for the synthesis of L-Tle and lays the foundation for large-scale production and application of chiral non-natural amino acids. |
topic |
leucine dehydrogenase trimethylpyruvic acid L-tert-leucine reductive amination amino acid |
url |
https://www.frontiersin.org/article/10.3389/fbioe.2020.00186/full |
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