Analysis of cholesterol oxidase activity and enzyme kinetics

ChoD activity was measured spectrophotometrically by the modified method of Sasaki et al. The stoichiometric formation of H₂O₂ during the oxidation reaction of cholesterol was monitored with ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) at 405 nm. To determine the cell-bound ChoD, cultures were centrifuged at 15,000 × g for 10 min. The cell pellet was resuspended in extraction buffer (0.15% Tween 80 in 50 mM phosphate buffer solution) and mixed for 30 minutes at 24 °C. The suspension was centrifuged at 15,000 × g and ChoD activity was measured from the supernatant. The activity assay mixture contained 120 μL Triton X-100 (0.05%) in 50 mM sodium-potassium phosphate buffer (pH 7), 10 μL ABTS (9.1 mM in MQ H₂O), 2.5 μL cholesterol in ethanol (1 mg/mL), 1.5 μL horseradish peroxidase solution (150 U/mL) and 20 μL of the extract preparation in a total volume of 154 μL. The spectrophotometric cholesterol activity assay was carried out in a 96-well plate. One unit of enzyme was defined as the amount of enzyme that forms 1 μmol of H₂O₂ per minute at pH 7.0 and 27 °C.

ChoD optimal activity for various pH levels was determined as above with only changes in the buffer as needed for specific pH tests as follows: 50 mM citrate buffer (pH 4–5), 50 mM potassium phosphate buffer (pH 6–7), and 50 mM Tris-HCl buffer (pH 8–9). For optimal temperature activity, temperatures between 25 and 75 °C were obtained using heat blocks. Each condition was tested in triplicates.

For analysis of enzyme kinetics, 8.4 nM ChoD was utilized to probe reaction velocities with eight substrate concentrations ranging between 4–168 µM cholesterol in triplicates. The initial rate of the reaction was calculated from derivatives of progression curves (six initial measurement points over 25 s) and referenced to a H₂O₂ standard curve.