Applied Microbiology and Biotechnology

, Volume 102, Issue 21, pp 9183–9192 | Cite as

Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase

  • Hong Yang
  • Yueqi Zhang
  • Xinxin Li
  • Yingguo Bai
  • Wei Xia
  • Rui Ma
  • Huiying Luo
  • Pengjun ShiEmail author
  • Bin YaoEmail author
Biotechnologically relevant enzymes and proteins


A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley β-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0–6.0 and 60–65 °C with barley β-glucan and CMC-Na as substrates and had a melting temperature (Tm) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and Tm values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.


Pichia pastoris Cellulase Disulfide bonds Thermostable Folding and refolding 



This study was supported by the National Key R&D Program of China (2016YFD050140902), the Chinese Academy of Agricultural Science and Technology Innovation Project (CAAS-XTCX201601101), and the National Chicken Industry Technology System of China (CARS-41).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9256_MOESM1_ESM.pdf (801 kb)
ESM 1 (PDF 801 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Hong Yang
    • 1
  • Yueqi Zhang
    • 1
  • Xinxin Li
    • 1
  • Yingguo Bai
    • 1
  • Wei Xia
    • 1
  • Rui Ma
    • 1
  • Huiying Luo
    • 1
  • Pengjun Shi
    • 1
    Email author
  • Bin Yao
    • 1
    Email author
  1. 1.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research InstituteChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China

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