DEVELOPMENT OF NEAR REAL-TIME MONITORING SYSTEMS FOR SOME SERINE PROTEASE ENZYMES IN THE INDUSTRIAL ATMOSPHERE

摘要

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.