Absorbance at 335 nm was measured immediately after adding substrate and then continuously for 20 minutes using a Cary 50 Microplate Reader in Nunc 96 well microtitre plates.
The reaction was initiated by adding substrate and incubated at 37°C for 60 min. Reaction was terminated by adding 200 µl of 0.6 N perchloric acid followed by centrifugation.
The capacity of indigenous microorganisms to degrade organic pollutants within an environment matrix can however be enhanced, either through stimulation by adding substrates and nutrients for example, or by the addition of contaminant-degrading microorganisms (Karlson et al. 1995; Sheehan 1997).
Parallel lines or lines intersecting at the y-axis for the second added substrate would be produced for either of these mechanisms, respectively.
Cells were washed 3 times with washing solution and added substrate solution and incubated for 3 10 min at room temperature.
After washing of unbound ACE, plate-bound ACE activity was measured by adding a substrate for ACE (Hip-His-Leu) directly into wells [33], [36].
This specific compound or protein can then be quantified by adding a substrate for the reporter enzyme and measuring the concentration of the product [ 22].
The in situ signal of the gene of interest was revealed by adding NBT/BCIP substrate for alkaline phosphotase reaction.
Subsequently succinate (5 mM) was added as substrate for mitochondrial ROS production, and digitonin (50 μg/ml) was added to permeabilize the cell membrane.
After this procedure, p-nitrophenyl phosphate (pNPP) was added as substrate for alkaline phosphatase following detection at 405 nm.
