Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b

The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances.Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin.Aerobic methanotrophic microorganisms are Gram-negative bacteria that have the unique ability to use methane as their only source of carbon and energy [1]. In a first step of their metabolic pathway, methanotrophs oxidize methane to methanol, a reaction catalyzed by methane monooxygenase (MMO). Whereas most aerobic methanotrophs produce a membrane-bound, or particulate MMO (pMMO), some strains also produce a cytoplasmic, or soluble MMO (sMMO) under copper-limiting conditions [1,2]. Both types of MMO contain metal atoms which are involved in the electron transfer to the substrate. pMMO contains several copper atoms [3,4] and possibly a diiron center [3], whereas sMMO holds a diiron active site [5,6]. Copper has been found to play a key role in the regulation of pMMO and sMMO expression as well as in the regulation of other metabolic enzymes of methanotrophs [4,5,7,8]. Furthermore, methane oxidation by pMMO critically depends on copper availability [9]. Due to their high copper requirement, methanotrophs necessitate an efficient copper mobilization and uptake system.Studies conducted in the 1990s provided the first evidence for the production o