Structure, function and engineering of thermostable GH-7 family cellobiohydrolases

Cellulases are important industrial enzymes, which can be used e.g. in the pulp and paper, textile, and detergent industry. They are currently under intensive study particularly due to their applicability in total hydrolysis of cellulosic biomass for production of bioethanol and other chemicals. Thermostable enzymes are desirable for many cellulase applications, as they are generally robust, tolerating various harsh process conditions. Thermophilic organisms are a potential source for thermostable enzymes, or as an alternative the thermostability of an enzyme can be improved by structure-based rational mutagenesis or directed evolution methods. Glycosyl hydrolase family 7 (GH-7; see http://afmb.cnrs-mrs.fr/CAZY/citing.html) cellobiohydrolases seem to be especially important in the hydrolysis of highly crystalline cellulose, and are found only in the fungal kingdom. Here, we present the characterisation of the enzymatic properties of three novel thermostable cellobiohydrolases originating from the thermophililic fungi Acremonium thermophilum, Chaetomium thermophilum and Thermoascus aurantiacus. These GH-7 family enzymes were expressed in the industrially relevant production host Trichoderma reesei, and the kinetics on small soluble substrates, cellobiose inhibition, crystalline cellulose hydrolysis and thermostability of the purified enzymes were determined. Enzymatic properties of the cellobiohydrolases were compared to those of T.reesei cellobiohydrolase Cel7A, one of the most thoroughly studied fungal cellobiohydrolases. In addition, both random and site-directed mutagenesis approaches have been used to improve the performance of the Melanocarpus albomyces Cel7B at elevated temperatures. The biochemical results are also discussed from the structural point of view based on the three-dimensional structures and homology models of these enzymes. Acknowledgements: This work has been supported by grants from the EU ("Technological improvement for ethanol production from lignocellulose" project; coordinator L. Viikari, VTT), the Academy of Finland (SV) and the Finnish Glycoscience Graduate School (SV). References : [1] Voutilainen, S., Boer, H., Linder, M., Puranen, T., Rouvinen, J., Vehmaanperä, J. and Koivula, A. (2007) Heterologous expression and random mutagenesis to improve the thermostability of Melanocarpus albomyces Cel7B cellobiohydrolase. Enz. Microb. Technol. 41, 234-243. [2] Voutilainen, S., Puranen, T., Siika-aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanperä, J. and Koivula, A. (2008) Cloning, expression and characterization of novel thermostable family 7 cellobiohydrolases. Biotechn. Bioeng.101, 515-528. [3] Parkkinen, T., Koivula, A., Vehmaanperä, J. and Rouvinen J. (2008) Crystal structure of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding. Protein Sci. 17, 1383-1394. [4] Voutilainen, S., Boer, H., Alapuranen, M., Jänis, J., Vehmaanperä, J. and Koivula, A. (2008) Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. - Submitted for publication.