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

摘要

Background: The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of thebiogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report apurification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phaseHPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of theresin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as wellas with elemental analysis and ESI-MS.Results: The results showed that numerous compounds other than methanobactin were present in the isolateobtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicatedthat methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carboncontributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results impliedthat besides methanobactin, the organic compounds in the resin extract comprised breakdown products ofmethanobactin as well as polysaccharide-like substances.Conclusion: Our results demonstrate that a purification step is indispensable in addition to resin extraction inorder to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparativework and provides copper-free methanobactin.