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High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production

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Abstract

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

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Acknowledgments

This work was financially supported by Tongsuh Petrochemical Corp., Ltd.

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Authors and Affiliations

  1. Department of Food Science & Biotechnology, Kyungsung University, 309, Suyeong-ro, Nam-gu, Busan, 608-736, South Korea

    Mi-Suk Kang & Jin-Ho Lee

  2. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Gusung-dong, Yusung-gu, Daejon, 305-701, South Korea

    Sang-Soo Han & Hak-Sung Kim

  3. Tongsuh Petrochemical Corp., Ltd., 154, Bugok-dong, Nam-gu, Ulsan, 680-110, South Korea

    Mi-Young Kim, Bu-Youn Kim & Jong-Pil Huh

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  1. Mi-Suk Kang
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  2. Sang-Soo Han
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Correspondence to Hak-Sung Kim or Jin-Ho Lee.

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Mi-Suk Kang and Sang-Soo Han equally contributed to this work.

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Kang, MS., Han, SS., Kim, MY. et al. High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production. Appl Microbiol Biotechnol 98, 4379–4387 (2014). https://doi.org/10.1007/s00253-014-5544-7

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  • DOI: https://doi.org/10.1007/s00253-014-5544-7

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