In this study the enzymatic activity of adsorbed Thermomonospora fusca E��� and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. In particular, cellulases were allowed to contact hydrophobic polystyrene surfaces under conditions of different solution concentrations, and adsorption times. Each of these variables is known to have a potential effect on enzyme structure and activity at an interface. Enzymatic activity was measured after partial elution of the adsorbed layer with both protein-free buffer and the surfactant, dodecyltrimethylammonium bromide. For E��� at high concentration (0.5 mg/ml), adsorbed enzyme activity decreased about 20% in increasing adsorption time from 0.25 h to 24 h. At low concentration (0.001 mg/ml), adsorbed enzyme activity decreased by one order of magnitude during a 24 h period. CBHI layers lost activity only after a sufficiently long contact time with the surface, and this effect was not strongly dependent on enzyme concentrations in solution. These findings were explained with reference to structural changes undergone by adsorbed enzyme as a function of time and available interfacial area. / Graduation date: 2000

Graduation date: 1999

In this research, the interfacial behavior of Trichoderma reesei CBHI and Thermomonospora fusca E��� cellulases were studied at synthetic surfaces. For this purpose, colloidal silica and polystyrene particles were used to prepare cellulase-particle suspensions that were analyzed by several solution-phase techniques. These included circular dichroism spectroscopy, size exclusion chromatography and filtration, and a spectrophotometric assay for cellulase activity. All techniques were performed in the presence and absence of particles. Circular dichroism spectroscopy (CD) and size exclusion chromatography showed, however, that binding did not occur between either cellulase and silica, presumably because silica is hydrophilic and negatively charged. Binding did occur between each cellulase and polystyrene, most likely mediated through hydrophobic associations. Cellulase-polystyrene complexes were not analyzed using CD because of high light absorption by the polystyrene nanoparticles. Upon adsorption to polystyrene, the activity of the E��� dropped about 95% relative to that of the free enzyme. While this substantial loss in activity may have been the result of binding being mediated through the catalytic domain, strong evidence supporting the thought that adsorption occurs through hydrophobic associations, mediated through the binding domain, suggests that structural or steric factors were partly responsible for the loss. / Graduation date: 1999