2.2. Biosensor Determination of 3-Chlorobenzoate-1,2-Dioxygenase Activity

The activity of 3-chlorobenzoate-1,2-dioxygenase was determined as the change in respiration of intact freshly harvested 3-CBA-grown cells of actinobacterium in response to the enzyme substrate (the rate of cells response to a substrate, i.e., the rate of enzymatic reaction) using the electrochemical reactor microbial sensor.

For determination of the activity of 3-chlorobenzoate-1,2-dioxygenase, 3-CBA-grown R. opacus 1 CP cells were separated by centrifugation (7000 g, 10 min, +4 °C), washed twice with 50-mM Tris-HCl, pH 8.0, suspended in the same buffer and immediately used for analysis.

The measurements were performed in an air-saturated 50-mM Tris-HCl buffer, with a pH of 8.0, at room temperature in a 5-mL open cell equipped with a mixer. When basal cell respiration level was stabilized, 3-CBA was injected into the cell and the rate of oxygen concentration change was measured using a Clark oxygen electrode. The recorded signal (the rate of cell response to 3-CBA) reflected the rate of the enzymatic reaction of 3-CBDO with the substrate. The rate unit was pA/s: 1 pA/s ~ 0.153 μg O₂/(L s).

The oxygen electrode was equipped with an “Ingold 531 O₂ amplifier” (Instrumentation Laboratory (MI), Milan, Italy). The signal was recorded with a two-coordinate “XY Recorder-4103” (Laboratorni Přistroje, Praha, Czech Republic).