Optimizing CjMQO Enzymatic Activity

The linearity range of CjMQO activity was evaluated using a dose-response curve following a method reported previously with several modifications (Hartuti et al., 2018 (link); Acharjee et al., 2021 (link)). The spectrophotometric assay was performed in triplicate at 37°C with a spectrophotometer (V760, Jasco Co., Tokyo, Japan) connected to an open bath circulator (Julabo ED, Julabo Labortechnik GmbH, Germany). The CjMQO concentrations ranged from 0.01 to 2 μg/mL in a 1 mL reaction mixture containing 50 mM HEPES (pH 7.0), 20 μM decylubiquinone (dUQ, Sigma-Aldrich Inc.), 1 mM potassium cyanide (KCN, Sigma-Aldrich Inc.) and 120 μM 2,6-dichlorophenolindophenol (DCIP, Sigma-Aldrich Inc.). The reaction was started by adding 10 mM sodium malate to the reaction mixture, and the reduction of DCIP was measured at 600 nm. The specific activity was calculated by using the extinction coefficient ( ${\epsilon }_{600}$ ) of DCIP of 21 mM^-1 cm^-1. The optimum temperature for the activity of purified CjMQO was determined in triplicates under different temperatures as indicated above at fixed concentration of 0.2 μg/mL of purified CjMQO.
The optimum pH was determined as described previously (Hartuti et al., 2018 (link); Acharjee et al., 2021 (link)) by measuring CjMQO activity at different pH values using 50 mM of HEPES-NaOH (pH 6.8–8.4), Tris-HCl (pH 6.9–9.0), sodium phosphate (NaPi, pH 5.8–8.0), potassium phosphate (KPi, pH 5.8–8.0), MOPS-NaOH (pH 6.5–8.9), and CHES-NaOH (pH 8.6–10.0) buffers at 37°C and 0.2 μg/mL of purified CjMQO with a multi-mode microplate reader (SpectraMax Paradigm, Molecular Devices, San Jose, CA, United States).