Most cellulose degrading enzymes have a two-domain structure consisting of a catalytic domain and a cellulose-binding domain (CBD). The domains form well defined units which are separated by a distinct linker region. The CBD mediates the binding of the enzyme to the solid cellulose substrate. It is not involved in the hydrolysis, but if it is removed, much of the enzymes activity towards cellulose is lost. Typical examples of CBD containing enzymes are the major cellulases from the fungus Trichoderma reesei. The type of CBDs found in T. reesei consist of about 40 amino acids. The most studied one is from the enzyme cellobiohydrolase I (CBHI). Its structure resembles a wedge with two distinct faces. In this work the focus has been on functional studies on this type of CBDs and in particular that of CBHI. It was shown that several amino acid residues on one of the faces of the wedge, the flat face, are involved in the binding to cellulose. Several modifications on the other face, the rough face, did not to markedly affect the binding of the CBD. This led to the conclusion that the participation of the rough face to cellulose binding is very improbable. It was also shown that CBDs from different enzymes can have different binding affinities. The difference in affinity between CBHI and the higher affinity CBD of endoglucanase I was shown to be largely dependent on only one amino acid difference on the binding face. The effect of linking two domains was studied by constructing a double CBD consisting of the cellobiohydrolase II (CBHII) at the N-terminus and the CBHI CBD at the C-terminus. The two domains were linked by a 24 amino acid long linker. It was found that the linkage in the double CBD led to a substantially higher affinity towards cellulose compared to either of the two individual domains by themselves. A mechanism to explain the affinity increase based on the two-domain structure is presented. It is also proposed that a similar mechanism might affect the interaction of the wild type enzymes with cellulose. Finally, the reversibility of the CBD binding to cellulose was investigated. This is an issue which has been unclear, and is intimately linked to the function of the enzyme and applications of CBDs. The CBHI CBD was radioactively labeled with tritium, which allowed more detailed studies of its behavior than were possible before. It was shown that if a sample with CBD and cellulose at equilibrium is diluted, then a new equilibrium further down the same isotherm is established. This result clearly shows that the binding is reversible. It was also shown that the CBHI CBD had an exchange rate at the surface which would not be rate limiting for a processive enzyme to move along the cellulose surface during hydrolysis.
|Award date||24 Jan 1997|
|Place of Publication||Espoo|
|Publication status||Published - 1996|
|MoE publication type||G5 Doctoral dissertation (article)|
- cellulose-binding domain