Sensitive plate assay for screening and detection of bacterial polyurethanase activity

Aims: A plate assay to screen and detect bacterial polyurethanase in agar medium containing a colloidal polyester-polyurethane and rhodamine B is presented. Methods and Results: Substrate hydrolysis causes the formation of orange fluorescent halos visible upon u.v. irradiation. The logarithm of polyurethanase activity from a purified polyurethanase protein is linearly correlated with the diameter of halos, thereby allowing quantification of polyurethanase activities ranging from 0.81 to 7.29 Units. Conclusions: The potential advantages of this system are in identification and recovery of viable polyurethanolytic bacteria and quantification of polyurethanase activity. Significance and Impact of the Study: These advantages are derived largely from the intense fluorescence observed due to the hydrolysis of substrate reacting with rhodamine B allowing for the use of low substrate concentrations and corresponding decrease in time required detecting low levels of enzyme activity.