Aggarwal, Amit2012-02-222012-02-222012-01https://hdl.handle.net/11299/121006University of Minnesota M.S. thesis. January 2012. Major:Biochemistry, Molecular Biology and Biophysics. Advisors: Lawrence P. Wackett & Ping Wang. 1 computer file (PDF); viii, 85 pages.Atrazine is the most widely used herbicide by corn and sorghum growers. Atrazine chlorohydrolase (AtzA) dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. In this study, we report the cloning and initial experiments for partial characterization of mutant AtzA (Colin Scott, Colin J. Jackson, Chris W. Coppin, et al. 2009. Catalytic Improvement and Evolution of Atrazine Chlorohydrolase. Appl. Environ. Microbiol. 75(7):2184-2191). A plasmid vector was designed for ease of purification of the enzyme with the help of a his-tag and to ensure high expression of soluble protein in recombinant E.coli. The specific activity and substrate specificity of the purified mutant AtzA was then compared to the wild type enzyme. It was observed from these initial studies that the mutant enzyme had somewhat similar characteristics to the wild type enzyme. Further, the current study also describes immobilization recombinant E. coli cells expressing the wild type atrazine chlorohydrolase in a silica/polymer porous gel. This novel recombinant enzyme-based method utilizes both adsorption and degradation to remove atrazine from water. A combination of silica nanoparticles (Ludox TM40), alkoxides, and an organic polymer were used to synthesize a porous gel. Gel curing temperatures of 23°C or 45°C were used to either maintain cell viability or to render the cells non-viable, respectively. The enzymatic activity of the encapsulated viable and nonviable cells was high and extremely stable over the time period analyzed. At room temperature, the encapsulated non-viable cells maintained a specific activity between (0.44 ± 0.06) μmol/g-min and (0.66 ± 0.12) μmol/g-min for up to 4 months, comparing well with free, viable cells specific activities (0.61 ± 0.04 μmol/g-min). Gels cured at 45°C had excellent structural rigidity and contained few viable cells, making these gels potentially compatible with water treatment facility applications.en-USBiochemistry, Molecular Biology and BiophysicsThesis or Dissertation