Date of Completion
enzyme polymer conjugates; stability; polymers; bioconjugates; biosensors
Challa V. Kumar
Rajeswari M. Kasi
Field of Study
Doctor of Philosophy
This dissertation focuses on the development of a simple, efficient, and versatile method for enzyme stabilization and extends it into a universal approach for stabilization of enzymes on solid porous supports such as cellulose so that they can be used to produce novel enzyme biomaterials for advanced biocatalysis and biosensing applications. The novelty of this work is in the development of a universal and simple method for the stabilization of any enzyme, which is currently not available. Many enzyme conjugation methods are either enzyme-specific, or they require complex chemistries that significantly inactivate the enzyme.
Enzymes were first conjugated to the polymer poly(acrylic acid) (PAA). Carbodiimide chemistry was used for all crosslinking purposes, where the primary amines from enzymes were conjugated to carboxylic acids on PAA. The PAA “armors” the enzyme and restricts the conformational entropy of the enzyme’s structure, thus making it difficult for the enzyme to denature or become inactivated by proteases and inhibitors. The studies provide a powerful tool for the development of novel enzyme-PAA based conjugates with enhanced stability.
We also developed a simple and inexpensive method for the immobilization of enzymes in filter paper. We crosslinked enzyme with PAA in filter paper, and found that the enzyme-polymer conjugate forms around the fibers of the paper. The resulting enzyme-PAA conjugate remained ‘locked-in’ place, and the enzymes were not washed off when immersed in a solution. Additionally, these enzymes were protected by the PAA, thus providing a useful tool for the development of stable paper-based enzyme devices for use at room temperature, which can be washed with full recovery of enzyme.
We also designed an approach with bovine serum albumin (BSA) as an alternative to PAA, interlocking it with enzymes in a one-pot two-layer approach. In our design, BSA passivates the surface of cellulose, while at the same time providing attachment groups for subsequent chemical modification in a second layer. Thus, this one-pot two-layer method provides enzymes with enhanced stability, increased recyclability, and high retention in activity. These approaches provide powerful tools for creating novel enzyme-based paper devices that require enhanced stability at room temperature.
Riccardi, Caterina, "NanoArmoring of Enzymes in Ordinary Paper" (2018). Doctoral Dissertations. 1828.