University of Minnesota
Melamine (2,4,6-triamino-1,3,5-triazine) has been used on numerous occasions by individuals as a food adulterant to boost the apparent nitrogen content of food. Together with cyanuric acid, melamine causes acute kidney failure. In the recent infant formula incident, 150,000 children were hospitalized and several died. The WHO recently requested the development of a rapid method to quantitatively detect melamine and related compounds in foods. Our laboratories at the University of Minnesota have collaborated with BIOO Scientific to develop and market an easy-to-use melamine test kit for foods (www.bti.umn.edu/gateway/melamine.html). The test kit is based on the enzyme melamine deaminase that was discovered at the University. Melamine deaminase liberates ammonia from melamine, which be detected via a simple color test. The toxicity of melamine has been shown in cats to be due to co-crystallization with cyanuric acid resulting in blockage of kidney tubules. There is now heightened awareness that melamine and cyanuric acid together in foods are highly toxic, and their cooccurrence in foods will cause deaths, disruption in food distribution systems, and a world-wide economic disaster. Thus, a rapid method to detect cyanuric acid in foods is urgently needed. Here we propose to develop a rapid test kit for detecting cyanuric acid in foods by purifying and stabilizing two enzymes, cyanuric acid hydrolase and biuret hydrolase, that act sequentially to liberate ammonia from cyanuric acid. If successful, we will collaborate with BIOO Scientific, which builds on our experience with enzymes involved in s-triazine metabolism and BIOOs expertise in food testing methodologies and marketing. We have previously purified cyanuric acid hydrolases so we have experience with this class of enzyme. Our laboratories have recently identified a highly stable cyanuric acid hydrolase that will be studied here to determine if it is suitable for use in a test kit. The gene encoding biuret hydrolase has been cloned into several different bacteria for testing. In the proposed studies, we will express biuret hydrolase in a recombinant E. coil bacterium to produce large quantities of enzyme. An alternative enzyme that has already been cloned and expressed, allophanate hydrolase, has been shown by us to hydrolyze biuret. Since the reaction rate is currently too slow for practical use, experiments will be conducted to increase the biuret hydrolysis rate of allophanate hydrolase. An advantage of using allophanate hydrolase is that it produces three molecules from one biuret molecule, resulting in increased sensitivity of the cyanuric acid test kit.
Analytic Methods Utilized: