The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activit y when interference by suspended biomass is present. Indoxyl is a highly fl uorescent intermediate in the conversion of indole to indigo by Tod. A fluo rescence-based assay was developed and applied to monitor Tod activity in w hole cells of Pseudomonas putida Fl biofilm from a continuously operated bi ofilter. Suspended growth studies with pure cultures indicated that indoxyl , as measured by fluorescence, correlated with indigo production (r(2) = 0. 89) as measured by spectrophotometry. Whole-cell enzyme activity was follow ed during growth on a minimal medium containing toluene. The maximum normal ized whole cell enzyme activity of 19+/-1.5 X 10(-4) mg indigo (mg protein) (-1) min(-1) was reached during early stationary phase. P. putida Fl cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enz yme activity of 5.0+/-0.2 X 10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions. (C) 2000 Published by Elsevier Science B.V. All rights reserved.