The food supply is vulnerable to chemical adulteration for economic gain or to inflict harm.  A recent example is the adulteration of milk with melamine. In China alone, 400,000 children were afflicted and the world was enlightened as to the vulnerability of the global food supply. Melamine could be detected in milk via chromatography and mass spectrometry but those methods did not provide for rapid testing of milk and other products at their source of production.  Our laboratory had identified the genes and enzymes involved in the bacterial metabolism of melamine. In vivo, the enzymes released ammonia and supported cell replication.  The enzyme initiating the metabolism, TriA, was explored for its potential use as part of a rapid, point-of-source test for melamine by analyzing for ammonia that is released in the enzyme-catalyzed reaction. The triA gene from Pseudomonas sp. NRRL B-12227 was cloned into Escherichia coli DH5a, expressed, and purified. The TriA protein was shown to catalyze the hydrolytic deamination of melamine to produce ammeline and ammonia. Steady-state kinetic determinations yielded a kcat/KM of 2 x 10-6 M-1s-1, a rate that would provide sufficient ammonia release at relatively low enzyme concentrations per test.  The substrate range was shown to be quite restricted, thus mitigating against enzyme-catalyzed false positives.  TriA production was scaled up in the BioTechnology Institute Pilot Plant to provide the enzyme to Bioo Scientific for the manufacture and marketing of a test kit to rapidly detect melamine in foods. The methods used for the melamine test kit are modular. TriA reacts exclusively with melamine to release ammonia and that, in turn, reacts with phenol and hypochlorite to produce indophenol that is intensely blue.  This general principle can be expanded to test for many other nitrogen-containing chemicals that reacts with the chemical of interest with the release of ammonia.

Biocatalyst-based detection of chemicals in foods

245th National Meeting (2013)

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