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High level extracellular production of a truncated alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli by the optimization of induction condition and fed-batch fermentation

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01–0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.

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Acknowledgments

Financial support from the National High Technology Research and Development Program of China (Grant 2014AA021302 and 2014AA021303) is gratefully acknowledged.

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Authors and Affiliations

  1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Hongchen Zheng, Zhenxiao Yu, Xiaoping Fu, Shufang Li, Jianyong Xu, Hui Song & Yanhe Ma

  2. Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China

    Hongchen Zheng, Zhenxiao Yu, Xiaoping Fu, Shufang Li, Jianyong Xu & Hui Song

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  1. Hongchen Zheng
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  2. Zhenxiao Yu
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  3. Xiaoping Fu
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  4. Shufang Li
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  5. Jianyong Xu
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  6. Hui Song
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  7. Yanhe Ma
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Correspondence to Hui Song.

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Zheng, H., Yu, Z., Fu, X. et al. High level extracellular production of a truncated alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli by the optimization of induction condition and fed-batch fermentation. J Ind Microbiol Biotechnol 43, 977–987 (2016). https://doi.org/10.1007/s10295-016-1773-3

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  • DOI: https://doi.org/10.1007/s10295-016-1773-3

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