Functional studies and engineering of family 1 carbohydrate-binding modules

The family 1 cellulose-binding modules (CBM1) form a group of small, stable carbohydratebinding proteins. These modules are essential for fungal cellulose degradation. This thesis describes both functional studies of the CBM1s as well as protein engineering of the modules for several objectives. The characteristics and specificity of CBM1s from the Trichoderma reesei Cel7A and Cel6A, along with several other wild type and mutated CBMs, were studied using binding experiments and transmission electron microscopy (TEM). Data from the binding studies confirmed that the presence of one tryptophan residue on the CBM1 binding face enhances its binding to crystalline cellulose. The two T. reesei CBM1s as well as the CBM3 from the Clostridium thermocellum CipA were investigated by TEM experiments. All three CBMs were found to bind in linear arrangements along the sides of the fibrils. Further analyses of the bound CBMs indicated that the CBMs bind to the exposed hydrophobic surfaces, the so called (200) crystalline face of Valonia cellulose crystals. The function and specificity of CBM1s as a part of an intact enzyme were studied by replacing the CBM from the exo-acting Cel7A by the CBM1 from the endoglucanase Cel7B. Apart from slightly improved affinity of the hybrid enzyme, the module exchange did not significantly influence the function of the Cel7A. This indicates that the two CBM1s are analogous in their binding properties and function during cellulose hydrolysis. The CBM1 was also used for immobilization studies. To improve heterologous expression of a CBM1-lipase fusion protein, a linker stability study was carried out in Pichia pastoris. A proline/threonine rich linker peptide was found to be stable for protein production in this host. For whole bacterial cell immobilization, the T. reesei Cel6A CBM1 was expressed on the surface of the gram-positive bacteria, Staphylococcus carnosus. The engineered S. carnosus cells were shown to bind to cellulose fibers. To exploit the stable CBM1 fold as a starting point for generating novel binders, a phage display library was constructed. Binding proteins against an amylase as well as against a metal ion were selected from the library. The amylase-binding proteins were found to bind and inhibit the target enzyme. The metal binding proteins selected from the library were cloned on the surface of the S. carnosus and clearly enhanced the metal binding ability of the engineered bacteria.