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Semi-Rational Engineering of Leucine Dehydrogenase for L-2-Aminobutyric Acid Production

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Abstract

L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD+ regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The “best” variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency (k cat/K M) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L-ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.

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Acknowledgements

This work was financially supported by the National Major Scientific and Technological Special Project for “Significant Scientific Instrument and Equipment Development” (2012YQ15008713), the Zhejiang Provincial Natural Science Foundation of China (Q17C050006), and for Zhejiang Provincial Public Welfare Technology Application Research Projects (2017C33157).

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Authors and Affiliations

  1. Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Jian-Miao Xu, Feng Cheng, Fang-Tian Fu, Hai-Feng Hu & Yu-Guo Zheng

  2. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Jian-Miao Xu, Feng Cheng, Fang-Tian Fu, Hai-Feng Hu & Yu-Guo Zheng

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  1. Jian-Miao Xu
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  2. Feng Cheng
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Correspondence to Yu-Guo Zheng.

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Xu, JM., Cheng, F., Fu, FT. et al. Semi-Rational Engineering of Leucine Dehydrogenase for L-2-Aminobutyric Acid Production. Appl Biochem Biotechnol 182, 898–909 (2017). https://doi.org/10.1007/s12010-016-2369-0

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