Date of Graduation

Summer 2020

Degree

Master of Science in Chemistry

Department

Chemistry

Committee Chair

Keiichi Yoshimatsu

Keywords

alkaline phosphatase, cellulose-binding domain, heavy metals, inhibition, plasmid, protein expression, pelB sequence

Subject Categories

Analytical Chemistry

Abstract

In recent years, paper-based microanalytical devices have attracted great attention in broad application areas including the colorimetric detection of heavy metal ions. The simplicity, portability, and low cost of the devices offer unique advantages over conventional instrumental analysis. This project utilizes the inhibition of an enzyme-catalyzed reaction by metal ions to develop a paper-based analytical device for the detection of heavy metal contaminants such as mercury. I selected alkaline phosphatase for this study. This enzyme possesses Mg and Zn ions in its active site. Therefore, the competitive displacement of these metal ions and/or denaturation of the enzyme by heavy metal ions would lead to the inhibition of the enzyme’s activity. I have investigated the effects of several metal ions at different concentrations on enzyme-catalyzed color development. To improve the sensitivity of the method, I envisioned to immobilize the enzyme onto the filter paper. An attempt to express alkaline phosphatase-cellulose binding domain fusion protein in E. coli did not yield successful results. However, I was able to express alkaline phosphatase fused with a pelB signal peptide (without cellulose-binding domain) in E. coli. This suggests that the pelB sequence may be contributing to the expression of this enzyme. This information provides an important insight for the preparation of alkaline phosphatase-cellulose binding domain fusion protein that can be applied to the development of a highly sensitive metal detection method in the future.

Copyright

© Adjoa Otubea Adams

Available for download on Monday, August 01, 2022

Open Access

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