Sensitive plate assay for screening and detection of bacterial polyurethanase activity

Aims: A plate assay to screen and detect bacterial polyurethanase in agar m edium containing a colloidal polyester-polyurethane and rhodamine B is pres ented. Methods and Results: Substrate hydrolysis causes the formation of orange fl uorescent halos visible upon u.v. irradiation. The logarithm of polyurethan ase activity from a purified polyurethanase protein is linearly correlated with the diameter of halos, thereby allowing quantification of polyurethana se 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 pol yurethanase activity. Significance and Impact of the Study: These advantages are derived largely from the intense fluorescence observed due to the hydrolysis of substrate r eacting with rhodamine B allowing for the use of low substrate concentratio ns and corresponding decrease in time required detecting low levels of enzy me activity.