Isolation of Extracellular Cobalt-free Corrinoid from Methanosarcina barkei

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Cobalt-free corrinoids (CFCs) were isolated from Methanosarcina barkei Fusaro cells growing on a methanol minimum medium. The methanogen cells excreted a trace of CFCs (9.1 μg/1) into the culture medium when cobalt-deficient methanol medium was used. Several CFCs were separated by column chromatographies on ion exchangers and paper electrophoresis, where a major CFC showed a similar characteristic to that of nucleotide-free corrinoid, Factor B (cobinamide), suggesting to be hydrogenobinamide. By chemical insertion of Co²⁺, Cu²⁺, and Zn²⁺ into CFCs, the corresponding corrinoid and its metal analogues were observed. Bioassay using Eschevichia coli 215 revealed that the major CFC (a yellow product obtained after alkaline treatment) and its copper and zinc analogues were inactive as cobalamin but were active as antimetabolites of cobalamin. However, the CFC greatly stimulated the cell growth of M. barkeri grown under cobalt-deficient conditions.