Development of near real-time monitoring systems for some serine protease enzymes in the industrial atmosphere

Rapid, sensitive and reusable assay systems have been developed which can be used for near real-time monitoring of some protease enzymes in the workplace. The systems are based on fluorescence measurement with flow analysis using a mini-bioreactor containing a chemically immobilized fluorophore-labelled protein substrate. Protease enzymes such as subtilisin digest the fluoro-substrate and release fluorescent fragments which are detected downstream by a fluorimeter. The assay systems were optimized and used for measurement of standard solutions of protease enzymes and enzyme-spiked non-biological detergent solutions. These exhibited satisfactory performance in terms of speed, sensitivity, reproducibility and linearity. Detection is fast with response times of 4–5 min. The relationship between the enzyme (subtilisin) concentration and the observed fluorescence signal is linear within the measured range (0–20 ng for a flow injection analysis system and 0–20 ng ml⁻¹ for a simulated air sampling open-loop system). For the latter system using a bioreactor with 0.9 ml immobilized matrix, the limit of detection at 95% confidence is 0.26 ng ml⁻¹. The signal is reproducible with s_r of 0.93% (n = 8) for continuous use over a 16 h period with repeated exposure to 2 ng ml⁻¹ subtilisin episodes.