V 330 spectrophotometer

From the fractions obtained during purification (cell-free extract, flow-through, wash, purified), 10 μL samples of 2 mg/mL protein were taken. After addition of SDS loading dye and incubation at 95 °C for 5 min, the samples were spun down at 13,000×g for 1 min, and then loaded onto an SDS-PAGE gel (GenScript, USA) and run according to the recommendations of the gel supplier. A protein ladder (PageRuler pre-stained, Thermo Fisher) was also loaded. The gels were run in a Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (Bio-Rad), and current was applied using a PowerPac^TM HC High-Current Power Supply (Bio-Rad), set at 120 V. When the blue front of the loading dye reached the bottom of the gel, the gel was removed from the chamber, rinsed with water and stained with Coomassie InstantBlue^TM (Fig. S1). Absorption spectra from 200 to 700 nm were taken of each purified fusion protein and diluted in buffer in a quartz cuvette (V-330 Spectrophotometer, JASCO). Using the obtained values at 280 and 441 nm, the protein concentration (ɛ₄₄₁ = 14.0 mM⁻¹ cm⁻¹) and FAD ratio could be calculated.

Activity was measured by monitoring the formation of NADPH at 340 nm. After mixing enzyme (≤0.1 μM) with substrate (cyclohexanol, if not stated: at 70 mM) in buffer (50 mM Tris-HCl pH 7.5), 100 μM NADP⁺ was added, briefly mixed in a cuvette, and then the reaction was followed (V-330 Spectrophotometer, JASCO). The slopes of the initial 20 s were used to determine the rates, in ΔAbs/min. This value was then divided by the extinction coefficient of NADPH (ɛ₃₄₀ = 6.22 mM⁻¹ cm⁻¹), in accordance with the Lambert-Beer law, resulting in a rate in mM/min. By dividing this value by the protein concentration in the reaction, the k_obs values were obtained. All measurements were done in technical duplicates or triplicates.