LAPSE:2023.2776
Published Article
LAPSE:2023.2776
High-Level Expression of Nitrile Hydratase in Escherichia coli for 2-Amino-2,3-Dimethylbutyramide Synthesis
February 21, 2023
Abstract
In the synthesis of imidazolinone herbicides, 2-Amino-2,3-dimethylbutyramide (ADBA) is an important intermedium. In this study, the recombinant production of nitrile hydratase (NHase) in Escherichia coli for ADBA synthesis was explored. A local library containing recombinant NHases from various sources was screened using a colorimetric method. NHase from Pseudonocardia thermophila JCM3095 was selected, fused with a His-tag and one-step purified. The enzymatic properties of recombinant NHase were studied and indicated robust thermal stability and inhibition of cyanide ions due to substrate degradation. After systematic optimization of fermentation conditions, the OD600 (optical density at 600 nm), enzyme activity and specific activity of recombinant strain E. coli BL21(DE3)/pET-28a+NHase reached 19.4, 3.72 U/mL and 1.04 U/mg protein at 42 h, representing 5.86-, 26.6- and 4-fold increases, respectively. These results offered an efficient recombinant whole-cell biocatalyst for ADBA synthesis.
In the synthesis of imidazolinone herbicides, 2-Amino-2,3-dimethylbutyramide (ADBA) is an important intermedium. In this study, the recombinant production of nitrile hydratase (NHase) in Escherichia coli for ADBA synthesis was explored. A local library containing recombinant NHases from various sources was screened using a colorimetric method. NHase from Pseudonocardia thermophila JCM3095 was selected, fused with a His-tag and one-step purified. The enzymatic properties of recombinant NHase were studied and indicated robust thermal stability and inhibition of cyanide ions due to substrate degradation. After systematic optimization of fermentation conditions, the OD600 (optical density at 600 nm), enzyme activity and specific activity of recombinant strain E. coli BL21(DE3)/pET-28a+NHase reached 19.4, 3.72 U/mL and 1.04 U/mg protein at 42 h, representing 5.86-, 26.6- and 4-fold increases, respectively. These results offered an efficient recombinant whole-cell biocatalyst for ADBA synthesis.
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Keywords
biocatalysis, imidazolinone herbicides, nitrile hydratase
Subject
Suggested Citation
Deng S, Zhu S, Zhang X, Sun X, Ma X, Su E. High-Level Expression of Nitrile Hydratase in Escherichia coli for 2-Amino-2,3-Dimethylbutyramide Synthesis. (2023). LAPSE:2023.2776
Author Affiliations
Deng S: Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
Zhu S: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Zhang X: Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
Sun X: Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
Ma X: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China [ORCID]
Su E: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China; Enzyme and Fermentation Technology Laboratory, College of Light Industry and Food Engineering, N [ORCID]
Zhu S: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
Zhang X: Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
Sun X: Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
Ma X: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China [ORCID]
Su E: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China; Enzyme and Fermentation Technology Laboratory, College of Light Industry and Food Engineering, N [ORCID]
Journal Name
Processes
Volume
10
Issue
3
First Page
544
Year
2022
Publication Date
2022-03-11
ISSN
2227-9717
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Original Submission
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PII: pr10030544, Publication Type: Journal Article
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LAPSE:2023.2776
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https://doi.org/10.3390/pr10030544
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Feb 21, 2023
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