Rapid and sensitive assay for some protease enzymes using a fluorosubstrate-immobilized bioreactor

Abstract

Protease enzymes are commonly used in industry; exposure of the workforce to these enzymes may lead to respiratory sensitization. Assays are therefore needed which can be coupled to air sampling devices to produce real-time monitoring systems which are sufficiently sensitive and rapid to provide an early warning system that indicates that the airborne concentrations of enzyme have exceeded occupational exposure limits. Current methods based on spectrophotometric end-points are too slow and insensitive for this application. An assay is reported that is based on the release of fluorescein covalently attached to porcine thyroglobulin which is itself immobilized onto glass beads in a bioreactor. Exposure of the bioreactor to the enzymes, subtilisin, trypsin, alcalase and esperase produces a fluorescent peak in a flow through cell within 3–4 min. The resulting signal is linear (r= 0.999, n= 5) over the range 2–50 ng for subtilisin and the reproducibility of the assay (s_r) at 5 ng is 0.8% for this enzyme.