|M.Sc Thesis||Department of Civil and Environmental Engineering|
|Supervisors:||Prof. Dosoretz Carlos|
|Assoc. Prof. Armon Robert|
The aim of this research was to study and characterize the in vitro conversion of atrazine to cyanuric acid by recombinant enzymes atrazine chlorohydrolase, hydroxyatrazine aminohydrolase and N-isopropylammelide N-isopropylaminohydrolase, entrapped in sol-gel glass.
The kinetic parameters of the enzymes were determined by Hanes plots and were found to be: Km=429.5 mmol Atr/l and Sac=0.704 mmol HA/mg protein-min for AtzA; Km=441.2 mmol HA/l and Sac=0.326 mmol N-iso/mg protein-min for AtzB; Km=1080.3 mmol N-iso/l and Sac=0.051 mmol CA/mg protein-min for AtzC.
Of the sol-gel glass procedures applied, employing sonication during hydrolysis with no addition of methanol, resulted the most efficient for Atz enzymes entrapment, with a relative remaining activity of nearly 42%. The use of stabilizing additives added to the enzyme solution prior to doping, considerably improved the recovered activity of the entrapped enzyme. Glycerol was found to be the best stabilizing additive among all the reagents tested, yielding a residual enzyme activity above 70%. The entrapment increased the temperature and pH stability of the enzymes compared to their free soluble form. The entrapped enzymes displayed good stability over time.
Following the determination of the optimal conditions for immobilized enzymes, experiments in continuous packed columns reactors were carried out. Three configurations of the columns (single, multiple and co-immobilized) packed with the entrapped enzymes, were investigated. Single column system was highly efficient in conversion of atrazine and its metabolites whereas the multiple column systems have been found to be more effective in atrazine transformation than the co-immobilized enzyme reactors.
Our results further indicate that the distinct kinetic properties among the individual enzymes exclude the possibility of co-entrapment whereas the use of a multiple-enzyme system in series with entrapped enzymes resulted in an effective transformation of atrazine to cyanuric acid.
Although further work still should be done to complete optimization, the findings presented highlight the potential of such a system for either monitoring or as a detoxification technology.