Purification and Characterization of MMOH

M. sporium strain 5 [American Type Culture Collection (ATCC) 35069] was cultured in nitrate mineral salt media (ATCC 1306) at 30°C until optical density at 655 nm reached 8 to 10 with a methane:air (v/v) ratio of 10 to 15%. The cells were harvested by centrifugation (11,300g) for 20 min at 4°C. The cell pellets were suspended in a lysis buffer containing Mops (25 mM), NaCl (25 mM), sodium thioglycolate (8 mM), l-cysteine (2 mM), (NH₄)₂Fe(SO₄)₂·6H₂O (200 μM), MgCl₂ (5 mM), deoxyribonuclease (DNase) (0.25 μl/ml), and phenylmethylsulfonyl fluoride (PMSF, 0.04 mg/ml) at pH 6.5. The cell suspension was sonicated at 4°C (CV334 model, Sonics), and the lysate was centrifuged at 30,000g for 45 min at 4°C. The supernatant was carefully decanted and filtered through a 0.22-μm membrane (Merck Millipore). The filtrate was loaded onto DEAE Sepharose Fast Flow, Superdex 200, and Q Sepharose Fast Flow columns attached to the ÄKTA Pure 25 L fast protein liquid chromatography system (GE Healthcare) to purify MMOH with >95% purity (30 (link)). The purified MMOH was applied to a ferrozine assay to monitor the iron-ferrozine complex (562 nm), and the results revealed 3.9 to 4.2 Fe/MMOH with coefficient of determination (R²) values >0.999 (14 (link)).