Characteristics and activity of SpmoB domain of particulate methane monooxygenase expressed in Escherichia coli BL21 (DE3)

Abstract Particulate methane monooxygenase is one of the methane monooxygenases of methanotrophic bacteria. The enzyme can convert methane to methanol in mild conditions. Cupredoxin domain recombinant protein (SpmoB) of particulate methane monooxygenase can oxidize methane to methanol. This protein was expressed in Escherichia coli BL21 (DE3) with Lac operon-based induction. SpmoB protein was isolated and refolded from the E. coli recombinant inclusion body. The pH and temperature dependence of SpmoB activity was also investigated to increase its activity. the SpmoB protein expressed by E. coli BL21 (DE3) is about 39 kDa. The SpmoB inclusion body was solubilized in 8 M urea followed by stepwise dialysis to get the active form SpmoB protein. The specific activity of the refolded protein was 0.46 methanol mg protein-1 min-1, which was higher than that of SpmoB from the previous study. The SpmoB was an acidic protein with the highest methanol production at pH six and a temperature of 30°C, which are higher than full-length pMMO. The SpmoB activity was stable at pH 6 to 8, and the Vmax and Km were 0.380 μM methanol s-1 and 44.27 μM, respectively.