The cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei. Interaction with cellulose and application in protein immobiliz: Dissertation

The enzymatic degradation of cellulose, the major organic carbon source in the biosphere, is essential for maintaining the global carbon cycle. The hydrolytic degradation is a complex process involving a number of cellulolytic enzymes which together are capable of solubilizing the inert and solid cellulosic substrates. The cellulase system of the soft rot fungus Trichoderma reesei is the most studied and best understood of all cellulolytic systems. Cloning of the genes of the major cellulases, detailed biochemical characterization and structure determination of two enzymes and has led to improved understanding of the catalytic mechanisms of T. reesei cellulases at the molecular level. In the present investigation, site directed mutagenesis was used to study the binding mechanisms of the major T. reesei cellobiohydrolase I, CBHI, to cellulose. The enzyme consists of two separate domains: a catalytic domain which is separated from a smaller cellulose binding domain (CBD) with a glycosylated linker peptide. On the basis of the three dimensional structure, specific mutations were introduced to the CBD. Four mutant proteins and the wild type CBHI and core protein were first expressed in a heterologous host organism, Saccharomyces cerevisiae. The proteins suffered from severe overglycosylation, which altered their enzymatic properties. Therefore three of the mutants were also expressed in the native fungal host organism. Examination of the properties of the mutants showed that the CBD interacts with cellulose with a conserved aromatic amino acid (Y492) located on the tip of the wedge shaped molecule. It was also shown that the flat and more hydrophilic surface of the CBD is functionally more important. On the basis of the structures of a cellulose crystal and the CBD it could be concluded that hydrogen bonding also has a critical role in the adsorption of the CBD to cellulose. It was further demonstrated that the adsorption is controlled by electrostatic interactions between the enzyme molecules. High ionic strength increased the binding and activity of the proteins, which suggests that a hydrophobic effect is involved in the adsorption. The binding experiments on cellulose indicated that the role of the CBD is to increase the number of binding sites on the cellulose surface whereas the binding of the core protein may be restricted only to free chain ends on crystal defects and at the ends of the crystals. The importance of the two domain structure was also examined by introducing specific deletions to the interdomain linker peptide. One third of the linker peptide could be deleted without loss in the catalytic activity. However, the adsorption was reduced, indicating that the mode of interaction was changed as a result of the mutation. Deletion of the entire linker resulted in clear reduction in the activity to a level comparable with that of the core protein. Thus, sufficient separation between the two domains of CBHI is needed for productive interaction through the CBD. The role of the linker peptide is to facilitate the independent binding of both domains and to allow co operative interaction between the domains in the breakdown of the cellulose crystal. The CBD of T. reesei CBHI is very homologous to other CBDs found in fungal enzymes. The CBDs of bacterial enzymes are three times larger in size and there is no sequence homology with the fungal CBDs. In one part of this work, the adsorption properties of the extensively studied bacterial CBD of exoglucanase xylanase Cex from Cellulomonas fimi was compared with the properties of the CBD of CBHI from T. reesei. Two novel fusion proteins with a single chain antibody capable of binding 2 phenyl oxazolone (OxscFv) were constructed. The binding properties of the fusion proteins demonstrated that the CBD of Cex binds to cellulose with slightly better affinity. The adsorption capacity of the CBD of Cex to cellulose was four to sixfold higher depending on the cellulose used. Both fusion proteins were stably immobilized to cellulose and desorbed only with strong denaturing agents such as urea, guanidium chloride or SDS. When immobilized on porous cellulose both fusion proteins exhibited similar efficiencies (_14 %) in binding the multivalent hapten ox37 BSA. The results demonstrate that the CBDs of CBHI and Cex can both be used as affinity tags to immobilize proteins to cellulose. The purification of cellulases is very difficult and contaminating activities have caused considerable discrepancies in the reported enzymatic properties of cellulases. It was shown in one part of this study that even minor contamination with an endoglucanase at a level of less than 0.5 % considerably affected the properties of a T. reesei cellobiohydrolase II (CBHII) preparation in the hydrolysis of barley beta glucan.