Assaying the activities of Thermomonospora fusca E 5 and Trichoderma reesei CBHI cellulase bound to polystyrene

Abstract

The enzymatic activities of adsorbed Thermomonospora fusca E 5 and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. 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 an 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 5 adsorbed from solution at high concentration (0.5 mg/ml), adsorbed enzyme activity decreased about 20% as adsorption time was increased from 0.25 to 24 h. Adsorbed from solution at low concentration (0.001 mg/ml), adsorbed E 5 activity decreased by an 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 the enzyme concentration in solution during adsorption. These findings were explained with reference to structural changes undergone by adsorbed enzyme as a function of time and available interfacial area.