Fungal thermostable cellobiohydrolases. Characterization and protein engineering studies: Dissertation

Cellulases are important industrial enzymes, and they are already today utilized for example in the pulp and paper and textile industries. Due to their application potential in so-called second generation bioethanol production, starting from lignocellulosic raw materials, cellulases are currently in the centre of attention. Especially cellobiohydrolases are the key enzymes in total hydrolysis of biomass based processes. The currently known cellobiohydrolases from GH family 7 are, however, not highly active under industrial conditions. In this work two different approaches were taken to improve the hydrolysis of crystalline cellulose. Firstly, thermotolerance and activity of two known fungal cellobiohydrolases from GH7 family was improved by protein engineering and secondly novel cellobiohydrolases were studied. Engineering of GH7 cellobiohydrolases has been hindered because of difficulties in heterologous expression in a bacterial or yeast host. In this study a functional yeast expression system was developed for two single-module cellobiohydrolases. This heterologous expression system enabled protein engineering by random mutagenesis and site-directed mutagenesis. Random mutagenesis, carried out with error-prone PCR and followed by functional screening with an automated, thermostability screening method, was used for improving the thermostability of Melanocarpus albomyces Cel7B (Ma Cel7B). The stability and activity of Ma Cel7B was further improved through structure-guided protein engineering by introducing an additional disulphide bridge and a carbohydrate binding module. Rational mutagenesis was also used for engineering Talaromyces emersonii Cel7A (Te Cel7A). Altogether five individual S-S bridges were introduced to improve the stability of Te Cel7A. Three out of these five single S-S mutants had a clearly improved thermostability. These mutations were combined in a triple mutant, which had significantly improved unfolding temperature (by 9°C) and ability to hydrolyse microcrystalline cellulose at 80°C. In addition to the mutagenesis studies of known cellobiohydrolases, we found novel GH-7 family cellobiohydrolases, which have high activity on insoluble polymeric substrates. Three family 7 cellobiohydrolases originating from thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus and Chaetomium thermophilum were characterized and compared to the widely studied Trichoderma reesei Cel7A enzyme. The comparison revealed that all these novel cellobiohydrolases are promising for application purposes, because they were more thermostable than T. reesei Cel7A and more active in the hydrolysis of microcrystalline cellulose (Avicel) at 45°C.