CHEMICAL STUDIES ON VITAMIN B₁₂ AND ITS RELATED COMPOUNDS

XI. FORMATION OF 5, 6-DIMETHYLBENZIMIDAZOLYL COBAMIDE COENZYME AND OTHER UNIDENTIFIED VITAMIN B₁₂ ANALOGUES DURING THERMOPHILIC METHANE FERMENTATION IN THE PRESENCE OF EXOGENOUSLY SUPPLIED

Abstract

The formation of DBCC and unidentified vitamin B₁₂ analogues from exogenously added cyanocobalamin was demonstrated during thermophilic methane fermentation. Twenty-four hours after addition of cyanocobalamin the incubation mixture was centrifuged. The precipitate consisting of bacterial cells and sludges was extracted with hot ethanol. Vitamin B₁₂-active substances in the cultural filtrate were adsorbed on active charcoal and eluted with 80% ethanol. Each of the ethanol solutions obtained from the precipitate and from the filtrate was concentrated in vacuo, purified by usual phenol treatment and passed through DEAE-cellulose colume. Elution was carried out successively with water, 0.1N acetate buffer (pH 4.5) and 1M NaCl-acetone mixture as a developing solvent.
By column chromatography of the partial purificate obtained from the precipitate an orange-red effluent fraction was obtained (DC-1), which exhibited the same behaviors as DBCC in microbiological test, absorption spectroscopy and paper electrophoresis.
It also showed the coenzyme activity in Abeles-Lee's intramolecular oxidationreduction system. From these findings the identity of the orange-red substance with DBCC was concluded and the yield was calculated as ca. 20% of the cyanocobalamin added. Besides DBCC, the presence of an unidentified vitamin B₁₂-active substance was found in the fractions DC-2 (eluted with acetate buffer) and DC-3 (eluted with NaCl-acetone mixture).
Vitamin B₁₂-active substances in the cultural filtrate were fractionated in DS-1 (eluted with water), DS-2 (eluted with acetate buffer) and DS-3 (eluted with NaCl-acetone mixture).
In DS-1, the presence of a small amount of DBCC together with hydroxocobalamin, cyanocobalamin and an unidentified yellow-orange substance was detected.
In fraction DS-2, another yellow to yellow-orange substance was contained which moved slightly to the cathode. Though the lability in light was analogous to that of cobamide coenzyme (6), it did not show the coenzyme activity in Abeles-Lee's system. The significance of DBCC contained in methane bacteria was discussed.