Cellulose hydrolysis by Trichoderma reesei cellulases: Studies on adsorption, sugar production and synergism of cellobiohydrolase I, II and endoglucanase II

University dissertation from Department of Biochemistry (S)

Abstract: Three major cellulases, cellobiohydrolase I and II (CBH I and II) and endoglucanase II (EG II) of Trichoderma reesei have been purified by ion-exchange chromatography in an FPLC system. Microcrystalline cellulose (Avicel) was hydrolysed by the single enzymes and by equimolar mixtures of CBH I-CBH II and CBH I-EG II. Enzyme adsorption was followed indirectly by selectively quantifying the enzymes in the supernatant by ion-exchange chromatography in an FPLC system. The (synergistic) production of small, soluble sugars (glucose, cellobiose and cellotriose) by the enzymes was followed by HPLC. A relatively high glucose to cellobiose ratio (1:10) produced by CBH I indicated that the enzyme is not perfectly processive, i.e. the attacked cellulose chain dissociates from the active site after 5-10 successive cleavages. Production of high amounts of small soluble sugars (which are not expected as products of typical endo-attack on solid substrate) by EG II indicated that the enzyme has some exo-activity and/or it acts in a (pseudo) processive way (the same chain is attacked several times, but the chain dissociates from the active site in between). Adsorption results showed that the enzymes compete for common binding sites on the substrate, but they have specific sites as well. A two site Langmuir isotherm was used to fit parameters (saturation level and distribution coefficient) to the experimental data. According to the adsorption isotherm results, EG II has the most binding sites on the substrate, while CBH I has the strongest capability to displace other cellulases. Initial substrate conversion was linearly proportional with the bound enzyme amount in case of the cellobiohydrolases, showing that the average activity of the bound enzyme molecules does not change with increasing saturation. For EG II the corresponding curve levelled off indicating that some easily hydrolysed portions of the substrate are being depleted or saturated at high enzyme concentration. Synergism between CBH I and EG II at 40 C was rather stable (degree of synergism, DS: 1.27-1.45) but the synergism was negligible at 4 C. This is explained by that at low temperature both enzymes probably hydrolyse some loose ends on the substrate, i.e. they do not cooperate. At high temperature, when those sites are depleted, EG II must attack other regions, and will create new ends on the substrate which can be attacked by the exo-acting cellobiohydrolase, hence synergism is seen. Synergism between CBH I and CBH II is explained by the potential of the cellobiohydrolases to disintegrate the substrate thus making it more accessible for each other. Such a mechanism could explain the observed increase in synergism with increasing saturation of the substrate by these enzymes.

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