Fungal cellulases: Study of hydrolytic properties of endoglucanases from Trichoderma reesei and Humicola insolens

Abstract: Cellulose is one of the most abundant biopolymers on earth. Several organisms degrade cellulose, one of the most studied is the fungus Trichoderma reesei which produces seven genetically different cellulases. In this thesis, a thorough study of the hydrolytic characteristics of the less studied endoglucanases Cel12A (EG III), Cel45A (EG V) and Cel61A (EG IV) of T. reesei is presented. Cel12A showed lower endoglucanase activity than the major endoglucanases Cel5A (EG II) and Cel7B (EG I), however, the endoglucanase activity for Cel12A was significantly higher than what was observed for Cel45A and Cel61A. The need for expressing Cel12A might be found in the low molecular mass, only 25 kDa compared to about 50 kDa for the major endoglucanases. A smaller enzyme might penetrate parts of a natural cellulosic substrate to which the major enzymes cannot access. Cel45A showed high activity against glucomannan and only a low endoglucanase activity. Thus, since no activity on mannan was observed for Cel45A, the hydrolytic properties indicates that this enzyme is a glucomannanase. For Cel61A, only a low endoglucanase activity was observed. A novel approach to characterise soluble cellulose derivatives is presented. The cellulose derivative, carboxymethyl cellulose (CMC), was hydrolysed by a single endoglucanase and the products were analysed with several techniques, in particular liquid chromatography and MALDI-TOF mass spectrometry. Endoglucanases of the fungi Humicola insolens and T. reesei were used. Since the substituents inhibit the hydrolysis by the enzymes, total hydrolysis was not achieved by any endoglucanase. Furthermore, some of the endoglucanases were able to hydrolyse the CMC to higher extent than others. Thus, it is possible to use pure endoglucanases in analysing cellulose derivatives. Cellulases are able to hydrolyse cellulose in co-operation, the enzymes act in synergy. When studying the hydrolysis of lignocellulose with the exoglucanase Cel7A (CBH I) in combination with the endoglucanase Cel5A several observations were made which did not fit the most accepted synergy model. Therefore, an extension to the endo-exo synergy model is presented. The concept is introduced that the observed synergy is due to removal of obstacles for the exoglucanase by the endoglucanase activity.