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Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation

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Abstract

Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD Ap and NTD-fused ntdSOD Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD Ap . To the best of our knowledge, Mn-rec ntdSOD Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD Ap and ntdSOD Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.

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Author notes

  1. These four authors have contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin, 300-457, China

    Mingchang Li, Shuyi Guo, Lin Zhu, Chunyan Yin & Wei Wang

  2. National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi, 214-122, China

    Xiaomin Li

  3. Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry of China, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300-071, China

    Quan Wang

  4. Tianjin Key Laboratory of Microbial Functional Genomics, TEDA, Tianjin, 300-457, China

    Wei Wang

Authors
  1. Mingchang Li
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  2. Shuyi Guo
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  3. Xiaomin Li
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  4. Quan Wang
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  5. Lin Zhu
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  6. Chunyan Yin
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  7. Wei Wang
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Correspondence to Wei Wang.

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Li, M., Guo, S., Li, X. et al. Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation. Biotechnol Bioproc E 22, 725–733 (2017). https://doi.org/10.1007/s12257-017-0243-8

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  • DOI: https://doi.org/10.1007/s12257-017-0243-8

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