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 activity when interference by suspended biomass is present. Indoxyl is a highly fl...

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Bibliographic Details
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
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Summary: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.
ISSN:0167-7012
1872-8359
DOI:10.1016/S0167-7012(00)00123-8