Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Sanni Voutilainen, Harry Boer, M. Alapuranen, J. Jänis, Jari Vehmaanperä, Anu Koivula (Corresponding Author)

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    53 Citations (Scopus)


    Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.
    Original languageEnglish
    Pages (from-to)261-272
    Number of pages12
    JournalApplied Microbiology and Biotechnology
    Issue number2
    Publication statusPublished - 2009
    MoE publication typeA1 Journal article-refereed


    • Cellulase
    • Cellulose
    • Protein engineering
    • Saccharomyces cerevisiae expression
    • Site-directed mutagenesis


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