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Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0–pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60–70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).

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Acknowledgments

Financial support from the National High Technology Research and Development Program of China (Grant 2013AA102106), Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT1166), the National Natural Science Fund (Grant 31,101,219) and China Postdoctoral Science Fund (Grant 2013M540076) are gratefully acknowledged.

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Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology, Education Ministry of China, Tianjin, 300457, China

    Hongchen Zheng, Yihan Liu, Yang Han & Fuping Lu

  2. National Engineering Laboratory for Industrial Enzymes (NELIE), Tianjin, 300457, China

    Mingzhe Sun & Fuping Lu

  3. Tianjin Key Laboratory of Industrial Microbiology, Tianjin, 300457, China

    Yihan Liu & Jianling Wang

  4. Industrial Microbiology Laboratory, College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China

    Hongchen Zheng, Yihan Liu, Mingzhe Sun, Yang Han, Jianling Wang & Fuping Lu

  5. Chinese Academy of Agricultural Engineering, Beijing, 100125, China

    Hongchen Zheng & Junshe Sun

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  1. Hongchen Zheng
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  2. Yihan Liu
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  3. Mingzhe Sun
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  4. Yang Han
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  5. Jianling Wang
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  6. Junshe Sun
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  7. Fuping Lu
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Corresponding author

Correspondence to Fuping Lu.

Additional information

H. Zheng and Y. Liu, who contributed equally to this work, are both nominated as the first author.

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Zheng, H., Liu, Y., Sun, M. et al. Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis. J Ind Microbiol Biotechnol 41, 153–162 (2014). https://doi.org/10.1007/s10295-013-1363-6

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  • DOI: https://doi.org/10.1007/s10295-013-1363-6

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