Title

Novel bioactive enzyme/DNA/inorganic nano-biocomposite materials

Date of Completion

January 2006

Keywords

Chemistry, Biochemistry|Chemistry, Inorganic

Degree

Ph.D.

Abstract

The long term goal of our current studies is to develop sufficient understanding of enzyme-solid interactions to predict and manipulate the properties of bound enzymes. We surmised that control of interactions between the solid support and the bound enzyme is essential to predict the behavior of the bound enzyme. Stabilization of the native state and destabilization of the denatured state of the bound enzyme, for example, is expected to improve enzyme stability. Minimization of interactions with the solid will also minimize the perturbation to the bound enzyme structure and enhance the retention of the native structure of the bound enzyme to a significant extent. In a similar manner, the control of the orientation of the bound enzyme is essential to maintain access to the active site and maintain a high level of activity for the bound enzyme. Specifically, the goal of this dissertation involves developing methodologies to improve the properties of free and bound proteins and enzymes. Such materials will be useful for designing novel bioactive nano-biocomposites. ^ To achieve this goal: (1) efficient immobilization of enzymes/proteins at novel layered inorganic surfaces were achieved, (2) the bound enzyme were fully characterized in the structural/activity and thermodynamic studies, and lastly (3) the protein-protein and protein-solid interactions were attenuated in a rational manner by (a) increasing the ionic strength, (b) using DNA as a scaffold for protein immobilization, and (c) manipulating the bound protein population distribution among a continuum of thermodynamic states by adding co-solutes such as urea. Urea destabilization of partially unfolded proteins and their conversion to well-folded proteins is a worthy goal. ^ Such systematic approaches not only improved the properties (structure, stability and activity) of free and bound proteins/enzymes but also opened new avenues for engineering more effective synthetic materials for the applications such as biocatalysis, therapeutics and gene delivery. For example, our recent studies have resulted in (a) the formation of novel bioactive nano-biocomposites, (b) stability and activity (up to 5-fold) improvement of bound proteins/enzymes, (c) development of a methodology to bind DNA to a negatively charged solid, and in specific cases, (d) urea-induced stabilization of proteins with potential application in liquid formulations. ^ The current research works at the interface of microfabrication, pharmaceutics and drug delivery (PDD), biochemistry, physical chemistry, inorganic chemistry and material science to elucidate the protein-solid interactions in detail. This work will provide a framework to engineer more effective synthetic materials and may have enormous technical impact in the areas of drug delivery and therapeutics, health care, food industry, nano-biosensors, optics, molecular electronics and proteomics. ^