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Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum

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Abstract

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4–5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

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Acknowledgments

This work was funded by National High Technology Research and Development Program of China (863 Program) and National Natural Science Foundation of China (30571246).

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Authors and Affiliations

  1. Department of Environmental Biology, Shandong Agricultural University, Tai’an, Shandong, 271018, People’s Republic of China

    Xiu-Juan Wang, Yan-Jie Peng, Li-Qing Zhang, An-Na Li & Duo-Chuan Li

  2. State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong, 271018, People’s Republic of China

    Xiu-Juan Wang

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  1. Xiu-Juan Wang
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Correspondence to Duo-Chuan Li.

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Wang, XJ., Peng, YJ., Zhang, LQ. et al. Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum . Appl Microbiol Biotechnol 95, 1469–1478 (2012). https://doi.org/10.1007/s00253-011-3799-9

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