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Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

Applied Microbiology and Biotechnology volume 98, pages 2991–3001 (2014)Cite this article

Abstract

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S–S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.

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Acknowledgments

Arja Kiema and Eila Leino are thanked for the skilful technical assistance. We also wish to thank Roal Oy, Finland, for providing the T. aurantiacus β-glucosidase and Chemtext, Italy, for providing the pre-treated A. donax material. This study was performed in NEMO, a project under EU’s seventh framework program, grant number 222699.

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Affiliations

  1. VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland

    Sanni P. Voutilainen, Susanna Nurmi-Rantala, Merja Penttilä & Anu Koivula

  2. Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, 00076 Aalto, Espoo, Finland

    Sanni P. Voutilainen

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  1. Sanni P. Voutilainen
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  2. Susanna Nurmi-Rantala
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  3. Merja Penttilä
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  4. Anu Koivula
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Correspondence to Anu Koivula.

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Voutilainen, S.P., Nurmi-Rantala, S., Penttilä, M. et al. Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae . Appl Microbiol Biotechnol 98, 2991–3001 (2014). https://doi.org/10.1007/s00253-013-5177-2

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  • DOI: https://doi.org/10.1007/s00253-013-5177-2

Keywords

  • Cellulase
  • Carbohydrate-binding module
  • Disulphide bridge
  • Saccharomyces cerevisiae
  • Talaromyces emersonii
  • Protein engineering
  • GH7
  • CBM1
  • CBM2
  • CBM3