Studies on the practical applications and mechanism of nitrilase catalyzed reactions

Nitriles are an economically important class of compounds in agriculture and the chemical industry. With the growing awareness of the toxicity of nitriles, and their potential threat to the environment, a variety of enzymes have been discovered which utilize nitriles as their sole substrates. The nitrilases catalyze the hydrolysis of a variety of aromatic and heteroaromatic nitriles to the corresponding carboxylic acids and ammonia. We have purified the nitrilase from Rhodococcus sp. (ATCC 39484), and found that the enzyme is susceptible to a variety of active site directed reagents, undergoing rapid, time-dependent inactivation by reagents specific for cysteine, histidine and arginine amino acid residues. Inactivation protection by substrate suggests that the modification reactions are occurring at or near the active site, so these residues may be important for catalysis. These studies are consistent with a mechanism for nitrilase-catalyzed hydrolysis which proceeds through the initial formation of a covalent thioimidate intermediate. We have subsequently synthesized authentic thioimidates as chemical models for the putative intermediate of nitrilase catalyzed reactions, and conducted a preliminary study of their aqueous chemistry utilizing carbon-13 NMR spectroscopy.; We have also investigated potential practical applications of the nitrilase reaction. We have developed an electrochemical biosensor for the on-site detection and quantitation of nitrile concentrations in aqueous solution. Nitrilase has been immobilized on the surface of an ammonia gas sensing electrode, where the analyte is the ammonia produced from the nitrilase-catalyzed hydrolysis of benzonitriles. We have prepared and studied the physical and kinetic characteristics of biotinylated nitrilase and successfully attached the modified enzyme onto the surface of a glassy carbon microelectrode by a biotin-avidin-biotin linking tether. The application of immobilized nitrilase as a reusable catalyst for the hydrolysis of nitriles under mild conditions has also been investigated.