Date of Completion
cellulase; model cellulose; kinetic modeling; surface kinetics; surface plasmon resonance; graphene; bovine serum albumin; catalase; cytochrome c; lysozyme; hemoglobin; glucose oxidase; urease; multi-layer graphene; protein adsorption; kinetics; thermodynamics
Dr. Yao Lin
Dr. Challa Vijay Kumar
Dr. Rajeswari M. Kasi
Field of Study
Doctor of Philosophy
Biofuels from renewable plant biomass offer an alternative strategy to replace fossil fuels. However, due to recalcitrant nature of cellulosic biomass, it is an extremely difficult to disintegrate cellulose into sugars by using enzymes. In nature, the fungal cellulase system such as Trichoderma reesei uses the cellulase cocktail that can efficiently degrade the plant biomass. However, the cooperative mechanism of the synergistic actions of multiple cellulases on cellulose remains to be elusive. Herein, we develop a surface plasmon resonance (SPR) based approach to quantify the enzymatic hydrolysis of cellulose by fungal cellulases, including both the processive (CBHI) and non-processive (EGII) cellulases. This method allows for the monitoring the enzymatic adsorption and surface reactions without the interference of the product inhibitions, and the generation of a large number of kinetic profiles from varying enzyme compositions and concentrations, under different environmental conditions. By analyzing the kinetic results, we develop the kinetic models that describe the processive and non-processive enzymes respectively, and eventually integrate them to elucidate the synergistic actions of the mixture of these two types of cellulases under a range of different compositions. The mechanistic study may shed lights on the further bioengineering of cellulases in order to produce economical cellulosic biofuels.
Aiming at following the cellulase actions on SPR sensors with even higher sensitivity, we investigated the graphene-coated SPR sensor as previous reports suggest the plasmonic coupling between gold layer and graphene may drastically enhance the SPR signals. We found, contrary to the previous literature, the graphene based SPR sensor did not show any significant inherent signal enhancement. However, this graphene SPR sensor does allow for the study on the binding kinetics of the protein-graphene interactions using a small library of model proteins with varying sizes and surface properties. This result may potentially be instructive for graphene based biosensing for detection of trace amount of biological entities.
Anuganti, Murali, "Developing Surface Plasmon Resonance (SPR) Based Methods for Quantitative Analysis of Enzymatic Surface Reactions and Biological Interactions" (2018). Doctoral Dissertations. 1845.
Available for download on Sunday, June 28, 2020