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

• Published in: Journal of microbiological methods 2000-04, Vol.40 (2), p.181-191
• Main Authors: Woo, Hae-jin, Sanseverino, John, Cox, Chris D., Robinson, Kevin G., Sayler, Gary S.
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biofilms; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; indigo; indole; Indoles; Microbiology; NAD - metabolism; Oxygenases - metabolism; Pseudomonas putida; Pseudomonas putida - enzymology; Pseudomonas putida F1; Space life sciences; Spectrometry, Fluorescence - methods; TCE cometabolism; Toluene dioxygenase
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(00)00123-8

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 activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r2=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19±1.5×10−4 mg indigo (mg protein)−1 min−1 was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0±0.2×10−4 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.