The use of a microbial enzyme in the development of a rapid enzyme-based assay for Paracetamol

Abstract

The work in this thesis describes the development of a rapid assay for serum paracetamol. The developed assay utilises the microbial enzyme aryl acylamide amidohydrolase (amidase) to degrade paracetamol. One of the reaction products is then coupled with a phenolic compound to form a coloured product. Three strains of bacteria were selected from soil on the basis of their ability to degrade paracetamol. Each was then characterised, allowing identification to generic level and beyond. After small-scale cultural studies, one isolate was grown on a pilot-scale and the amidase extracted and purified. The activity of the enzyme was investigated and its characteristics determined. A product of enzyme-catalysed paracetamol degradation was determined as para-aminophenol. Various properties of this compound were examined. A reaction system was developed whereby para-aminophenol was coupled with ortho-cresol to form an indophenol dye. The reaction was carried out in the presence of a transition metal capable of acting as an oxidising agent. After formulation of a suitable method protocol linking the enzyme and colour reactions, the assay was investigated with regard to accuracy, precision, reproducibility and interference. The procedure compared favourably to other methods in use for paracetamol estimation and its reliability was confirmed by a clinical trial carried out under hospital conditions.

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