Comparative Biochemical Investigations of Cellobiohydrolase (Cel7A) of Trichoderma reesei: Effects of Loop Structure and Functions

Utilization of fossil resources has revolutionized the modern society, however large quantities of CO2 has been emitted and still is as a consequence of how we live. The growing amount of greenhouse gasses leads to global temperature increase, which has already led to pronounced impacts on the climate. Therefore, conversion from fossil sources to renewable energy is crucial, if we want to protect ourselves and the Earth from immense consequences. One of many solutions to the pending challenge, is to replace fossil fuels with bioethanol produced preferable from lignocellulosic materials. Lignocellulose is recalcitrant and therefore difficult to degrade, but some organisms, such as Trichoderma reesei, are able to do so, by secretion of enzymatic cocktails. Some of these are called cellulases, and of them the cellobiohydrolase I (CBH I) also known as Cel7A, is the most abundant. This is also a significant component of the industrial cocktail, and as the enzymes constitute a considerable amount of production costs, engineering for optimization at industrial relevant temperatures is much needed.

This dissertation mainly revolves around the Cel7A of T. reesei (mainly referred to as TrCel7A) with focus on the properties of the loops and the kinetics of it. The enzyme has a characteristic fold with eight peripheral loops (A1-A4 and B1-B4) forming the roof of the catalytic binding tunnel. Spanning this are four almost completely conserved tryptophans. We developed a method, utilizing the intrinsic tryptophan fluorescence to measure the complexation between the enzyme and the substrate in the tunnel. As the enzyme gets filled with substrate, water is excluded, and the fluorescence signal increases, as water is a quencher of fluorescence. By singularly mutating the tryptophans to alanine, we found that removal of the tryptophans had surprisingly little influence on the rate of complexation.

We also investigated the loops of this enzyme. TrCel7A and the related endoglucanase (Cel7B) are structurally similar, except from four heavily truncated loops that might reason the functional disparity between the two enzymes. We deleted the loops unique to TrCel7A singularly, followed upon several kinetic investigations. The results showed that the B2 loop was the key determinant for the kinetic characteristics of the CBH/TrCel7A. These findings were in line with another study, where we further investigated the B2 loop of TrCel7A. By site saturation of the N200 position, we found that deletion of the asparagine, or substitution to a small amino acid, likewise shifted the kinetic parameters to resemble those of an EG.

Finally, we also examined how TrCel7A was affected by pH and temperature in presence of insoluble substrate. We found that TrCel7A was unaffected to the surrounding pH from 2 - 5.5, if the activity was tested at a temperature where the enzyme was stable at the given pH. This was in contrast to pH-profiles made on the basis of soluble substrate hydrolysis, of which a classical bell shaped curve appeared. Furthermore, this was in contrast to pH-profiles of TrCel7B with the more open binding cleft. It is natural to think that the accessibility to the catalytic binding site has an influence on this difference, and that this is governed by the truncated loop structure.

Together, this work contributes to the understanding of the molecular mechanism of this industrially relevant enzyme, TrCel7A. We elucidated enzyme-substrate complexation rates, stability, pH effects and kinetics of several of the important loops as well as identified the key loop for TrCel7A properties. These findings may be applicable for future enzyme engineering and improvement of the industrially process for bioethanol production.