There is an increasing need to test for chemicals present in adulterated foods or food products. There is also an increasing need to degrade chemicals present in water. The s-triazine compounds compose a major class of industrial chemicals. The research in our labs focuses on developing microbial enzyme-based bioassays for s-triazines and understanding the enzymatic biodegradation of them. Cyanuric acid is an intermediate in bacterial metabolism of common s-triazine compounds. In 2008, intentional adulteration of milk and milk products (e.g., infant formula) with the s-triazine melamine affected 300,000 infants & youth in China, resulting in 50,000 hospitalizations and 6 deaths. In 2010, tons of melamine-tainted milk products were again for sale. Melamine is sometimes co-contaminated with cyanuric acid. When ingested together, they form insoluble crystals in kidneys, which may lead to renal failure. This scenario caused >13,000 pet deaths in the U.S. (2007) due to adulterated pet food. There is interest in detecting cyanuric acid and melamine, individually or together, in foods. With regards to s-triazines in water, cyanuric acid is a stable compound that accumulates over time in swimming pools disinfected with N-chloroisocyanuric acids. With high concentrations of cyanuric acid, disinfection is ineffective and public health is not protected from disease-causing microorganisms. To address this concern, the pool is drained, refilled with fresh water, and treated again.The research here identifies and characterizes microbial enzymes involved in s-triazine metabolism that could be used to 1) develop sensitive bioassays for cyanuric acid and/or 2) biodegrade cyanuric acid in water. Cyanuric acid hydrolase acts on cyanuric acid. Using 13C nuclear magnetic resonance (NMR) and mass spectrometry (MS), this research establishes the product of this reaction is carboxybiuret. This work also demonstrates carboxybiuret spontaneously decarboxylates to generate biuret. In the subsequent reaction, biuret hydrolase acts on biuret. Using an interdisciplinary approach combining bioinformatics, microbiology, molecular biology, protein separations, enzymology, quantitative analyses, 13C NMR, and MS, this research identifies and characterizes the first biuret hydrolase to be purified in active form (from R. leguminosarum bv. viciae 3841). Together, cyanuric acid and biuret hydrolases release ammonia, which is detected via the colorimetric Berthelot Assay. Cyanuric acid hydrolase belongs to rare family of unstable proteins. In an effort to identify a stable protein scaffold that could be modified to hydrolyze cyanuric acid, an array of amidohydrolase family members were analyzed for low-level activity with cyanuric acid to use as a starting point. Guanine deaminase from Bradyrhizobium diazoefficiens USDA110 was observed to have such activity. This is the only known metallo-enzyme to hydrolyze a ring-opening amide bond of cyanuric acid.
University of Minnesota Ph.D. dissertation. August 2013. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Dr. Lawrence Wackett. 1 computer file (PDF); xxix, 295 pages, appendices 1-5.
Cameron, Stephan Mary.
Biuret hydrolase and cyanuric acid hydrolase: enzymes for metabolism and detection of s-Triazines.
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