Purification of Pyridoxamine and Pyridoxamine 5´-Phosphate from Culture Broth of Rhizobium sp. 6.1C1
Keywords:Rhizobium sp. 6-1C1, purification, pyridoxamine, pyridoxamine 5´-phosphate, agar diffusion assay
Vitamin B6 is an essential micronutrient for the metabolism of proteins, lipids and carbohydrates. Recently, pyridoxamine shows a potent antioxidant for cellular well-being. The Gram negative bacterium, Rhizobium sp. 6.1C1 produces extracellular pyridoxamine (PM) and pyridoxamine 5´-phosphate (PMP) in synthetic media. In this work, PM and PMP from the cultured broth of R. 6.1C1 were purified with strong acid cation exchange chromatography (DOWEX 50W ´ 8). The amount of vitamin B6 was measured by agar diffusion assay using S. carlsbergensis TISTR 5345 (ATCC 9080) as the test organism. The elution profile showed 7 peaks (P1-P7) which responded to the growth of S. carlsbergensis. To determine the forms of vitamin B6, each peak was examined by reversed-phase HPLC. The retention time of P5 and P7 corresponded to that of PMP and PM standards, respectively. The retention time of P1-P4 and P6 corresponded to that of the synthetic medium. Both fluorescence spectra and dephosphorylation confirmed that P5 and P7 were PMP and PM, respectively. This purification method can be used as a model for the development of large scale purification of vitamin B6 from fermentation broth.
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