Aims: Particulate methane monooxygenase (pMMO) is an integral membrane protein that converts methane to methanol as the first step in the metabolic pathway of methanotroph bacteria. Methanotroph have a slow growth rate that make researcher have to develop an alternative approach by expressing the pMMO genes in Escherichia coli. However, it was very difficult to express all the pMMO encoded genes in E. coli and it is suspected that the protein might be toxic to E. coli. Therefore, this research tried another approach by expressing the active site of pMMO enzyme; cupredoxin domain of pmoB subunit encoded by spmoB gene. Methodology and results: The spmoB gene from Methylococcus capsulatus (Bath) was expressed in E. coli BL21 (DE3) under T7 promoter and pET15b as the expression vector. Several modifications were made so this gene would be expressed in the cytoplasm. Expression analysis with SDS-PAGE showed that overexpression of this gene could be done at several concentrations of IPTG and incubation temperature. The spmoB gene expression produced a recombinant protein with a size approximately 38.9 kDa. Assay of spmoB protein activity showed that the amount of methanol accumulated during methane oxidation by the recombinant strain was 0.114 mmol/mL culture.h. Conclusion, significance and impact study: We successfully expressed spmoB gene in E. coli BL21 (DE3) without high production of toxic compounds and it has methane oxidation activity. This result allowed further characterization of its potential applications.
Escherichia coli