Purification of Pyridoxamine and Pyridoxamine 5´-Phosphate from Culture Broth of Rhizobium sp. 6.1C1

Anutida SANGSAI; Panawan MOOSOPHON; Yanee TRONGPANICH

Authors

Anutida SANGSAI Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002
Panawan MOOSOPHON Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002
Yanee TRONGPANICH Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002

Keywords:

Rhizobium sp. 6-1C1, purification, pyridoxamine, pyridoxamine 5´-phosphate, agar diffusion assay

Abstract

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.

Downloads

Download data is not yet available.

Metrics

Metrics Loading ...

References

H Hellmann and S Mooney. Vitamin B6: A molecule for human health? Molecules 2010; 15, 442-59.

ML di Salvo, R Contestabile and MK Safo. Vitamin B6 salvage enzymes: Mechanism, structure and regulation. Biochim. Biophys. Acta 2011; 1814, 1597-608.

P Voziyan and B Hudson. Pyridoxamine the many virtues of a maillard reaction inhibitor. Ann. N. Y. Acad. Sci. 2005; 1043, 807-16.

S Chetyrkin, M Mathis, WH McDonald, X Shackelford, B Hudson and P Voziyan. Pyridoxamine protects protein backbone from oxidative fragmentation. Biochem. Bioph. Res. Co. 2011; 411, 574-9.

S Huang, J Zhang, L Wang and L Huang. Effect of abiotic stress on the abundance of different vitamin B6 vitamers in tobacco plants. Plant. Physiol. Biochem. 2013; 66, 63-7.

M Tazoe, K Ichikawa and T Hoshino. Production of vitamin B6 in Rhizobium. Biosci. Biotech. Bioch. 1999; 63, 1378-82.

M Ishida and K Shimura. Vitamin B-6 production with a cell-suspension of Achomobacter cycloclaster. Agr. Biol. Chem. 1970; 34, 327-34.

Y Tani and K Ogata. Studies on vitamin B6 metabolism in microorganism part XI. Extracellular formation of vitamin B6 by marine and terrestrial microorganism and its control. Agr. Biol. Chem. 1972; 36, 189-97.

BR Belitsky. Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5'-phosphate biosynthesis. J. Bacteriol. 2004; 186, 1191-6.

C Anutrakunchai, S Niamsanit, PP Wangsomnuk and Y Trongpanich. Isolation and characterization of vitamin B6 producing thermophilic bacterium, Geobacillus sp. H6a. J. Gen. Appl. Microbiol. 2010; 56, 273-9.

T Hoshino, Y Nagahashi, K Ichikawa and M Tazoe. 2008, Process for Producing Vitamin B6, U.S. Patent 7,371,547.

Y Trongpanich, S Phimwapi, S Niamsanit, P Wangsomnuk, M Boonmee and S Siri. Isolation and characterization of bacteria capable of producing pyridoxamine (PM) and pyridoxamine 5´-phosphate (PMP), vitamin B6 compounds. J. Gen. Appl. Microbiol. 2007; 53, 295-9.

L Lumeng, A Lui and TK Li. Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism. J. Clin. Invest. 1980; 66, 688-93.

L Atkin, AS Schultz, WL Williams and CN Frey. Yeast microbiological methods for determination of vitamins pyridoxine. Ind. Eng. Chem. Anal. Ed. 1943; 15, 141-4.

AOAC official method 961.15. Vitamin B6 (Pyridoxine, Pyridoxal, Pyridoxamine) in Food Extracts. Microbiologica Method. Final Action 1975. In: H Indyk and E Konings (eds.). Official Methods of Analysis of AOAC International. 17th ed. AOAC International, Gaithersburg, MD, 2000, p. 45-55.

MM Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72, 248-54.

CJ Agrouderlis. Simple high-performance liquid chromatographic method for the determination of all seven vitamin B6 -related compounds. J. Chromatogr. A 1997; 790, 83-91.

JJ Nevado, JA Pulgarin and MA Laguna. Simultaneous determination of pyridoxal and pyridoxamine by different spectrofluorimetric techniques. Talanta 1995; 42, 129-36.

V Ollilainen. HPLC analysis of vitamin B6 in foods. Agr. Food. Sci. Finland 1999; 8, 515-619.

T Sivaraman, TK Kumar, G Jayaraman and C Yu. The mechanism of 2,2,2 trichloroacetic acid-induced protein precipitation. J. Protein. Chem. 1997; 16, 291-7.

M van der Ham, M Albersen, TJ de Koning, G Visser, A Middendorp, M. Bosma, NM Verhoeven-Duif and MGM de Sain-van der Velden, Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography–tandem mass spectrometry. Anal. Chim. Acta 2012; 712, 108-14.

MJ Esteve, R Farré, A Frigola and JM Garcia-Cantabella. Determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in pork meat and pork meat products by liquid chromatography. J. Chromatogr. A 1998; 795, 383-7.

C Bueno and MV Encinas. Photophysical and photochemical studies of pyridoxamine. Helv. Chim. Acta 2003; 86, 3363-75.