Effects of Microbacterium laevaniformans Levans Molecular Weight on Cytotoxicity

  • Oh, Im-Kyung (Department of Food and Nutrition, Hanyang University) ;
  • Yoo, Sang-Ho (Department of Food Science and Technology, Sejong University) ;
  • Bae, In-Young (Department of Food and Nutrition, Hanyang University) ;
  • Cha, Jae-Ho (Department of Microbiology, Pusan National University) ;
  • Lee, Hyeon-Gyu (Department of Food and Nutrition, Hanyang University)
  • Published : 2004.10.01

Abstract

Levans produced from Microbacterium laevaniformans were isolated, characterized, and fractionated by molecular weight. TLC, HPLC, and GC-MS analyses of the exopolysaccharide showed that it was a fructan-type polymer and was composed of (2,6)- and (2,1)-glycosidic linkages. $^{13}C$-NMR analysis proved that the polysaccharide was mainly a $\beta$-(2,6)-linked levan-type polysaccharide. To investigate the cytotoxicity of the acetone-precipitated levan fractions such as M1, M2, and M3, HepG2, P388D1, U937, SNU-1, and SNUC2A cell lines were screened. Among the cell lines tested, the cytotoxicity of M1- M3 fractions were detected from only SNU-1 and HepG2 cells at the dosage level of $100-800\mu\textrm{g}ml$. The M2 fraction M_r$, 80,000) at 400 $mu{g/ml}$ had the greatest cell growth inhibition (84.6%) on SNU-1, while the M1 $(M_r$, 50,000) at $800\mu\textrm{g}ml$ showed the greatest (46.32%) on HepG2. To obtain more uniform M_r$ fractions of levan, the levan was further fractionated from S1 $(M_r$ 1,000,000) to S5 $(M_r$ 10,000) using gel permeation chromatography. Again, the S1-S5 fractions had strong cytotoxicity on SNU-1 and HepG2 cell lines. The greatest inhibition effects of S4 $(M_r$ 80,000) on SNU-1 and S5 $(M_r$ 10,000) on HepG2 were shown to be 49.5% and 73.0%, respectively. The cytotoxicity of the levan fractions was more effective on SNU-1 than on HepG2. Although the relationship between the Mw and the cytotoxicity was not clear, smaller $M_r$, fractions of levan showed greater growth inhibition effect on the cancer cell lines in general. Therefore, it was indicated that a specific Mw class of levan is responsible for the effective cytotoxicity.

Keywords

References

  1. Allen, P.Z. and E. A. Kabat. 1957. Studies on the capacity of some polysaccharides to elicit antibody formation to man. J. Exptl. Med. 105: 383- 394.
  2. Allen, P. Z. and W. H. Bowen. 1990. Immunochemical studies on levans from several strains of Actinomyces viscosus. Archs. Oral Bioi. 35: 35- 62.
  3. Calazans, G. M. T., C. E. Lopes, F. P. Francisca, and R. C. Lima. 1997. Antitumour activities of levans produced by Zymomonas mobilis strains. Biotechnol. Lett. 19: 19- 21.
  4. Calazans, G. M. T., R. C. Lima, F. P. Francisca, and C. E. Lopes. 2000. Molecular weight and antitumor activity of Zymomonas mobilis. Int. J. Biol. Macromol. 27: 245- 247.
  5. Carmichael, J., W. G. Degraff, A. F. Gazdar, J. D. Minna, and J. B. Michell. 1987. Evaluation of a tetrazolium based semiautomated colorimetric assay, assessment of chemosensitivity testing. Cancer Res. 47: 936- 940.
  6. Euzenat, O., A. Guibert, and D. Combes. 1997. Production of fructo-oligosaccharides by levansucrase from Bacillus subtilis C4. Proc. Biochem. 32: 237- 243. https://doi.org/10.1016/S0032-9592(96)00058-1
  7. Feingold, D. S. and M. Gehata. 1957. The structure and property of levan, a polymer of d-fructose produced by cultures and cell-free extract of Aerobacter levanicum. J. Poly. Sci. 22: 783- 790.
  8. Hakomori, S. J. 1964. A rapid permethylation of glycolipid polysaccharide catalyzed by methyl sulfonyl carbon ion dimethyl sulfoxide. Biochemistry 55: 205- 208.
  9. Han, Y. W. 1990. Microbial levan. Adv. Appl. Microbiol. 35: 171-194. https://doi.org/10.1016/S0065-2164(08)70244-2
  10. Han, Y. W. and M. A. Clarke. 1990. Production and characterization of microbial levan. J. Agric. Food Chem. 38: 393- 396.
  11. Han, Y. W. and M. A. Watson. 1992. Production of microbial levan from sucrose, sugarcane juice, and beet molasses. J. Ind. Microbiol. 9: 257-260.
  12. Jang, K-H., S. A. Kang, Y. Cho, Y.-Y. Kim, Y.-J. Lee, K. Hong, K.-H. Seong, S. H. Kim, C.-H. Kim, S.-K Rhee, S.-D. Ha, and R. Choue. 2003. Prebiotic properties of levan in rats. J. Microbiol. Biotech. 13: 348- 353.
  13. Johnes M. R., P. F. Greenfield, and H. W. Dolle. 1991. By-products from Zymomonas mobilis. Adv. Biochem. Eng. Biotechnol. 44: 97-- 101.
  14. Kim, H., H. Park, M. Kim, H. G. Lee, J. Yang, and J. Chao 2003. Enzymaticcharacterization of a recombinantlevansucrase from Rahnella aquatilis ATCC 15552. J. Microbiol. Biotech. 13: 230- 235.
  15. Leibovici, J., S. Kopel, A. Siegel, and O. Gal-Mor. 1986. Effect of tumor inhibitory and stimulatory doses of levan, alone and in combination with cyclophosphamide, on spleen and lymph nodes. Int. J. Immunopharmacol. 8: 391- 403.
  16. Newbrun, E. and S. Baker. 1968. Physico-chemical characteristics of the levan produced by Streptococcus salivarius. Carbohydr: Res. 6: 165- 170.
  17. Otterlei, M., A. Sundan, G. Skjak-Braek, L. Ryan, O. Smidsrod, and T. Espevik. 1993. Similar mechanisms of action of defined polysaccharides and lipopolysaccharides: Characterization of binding and tumor necrosis factor alpha induction. Infect. lmmun. 61: 1917- 1925.
  18. Rolf, D. and G. Gray. 1982. Analysis of the linkage positions in D-fructofuranosyl residues by the reductive cleavage method. J. Am. Chem. Soc. 104: 3539-3541.
  19. Rosell, K. G. and D. Birkhed. 1974. An inulin-like fructan produced by Streptococcus mutans, strain JC2. ACTA Chem. Scand. 28: 589-592.
  20. Simms, P. J., W. J. Boyko, and J. R. Edwards, 1990. The structure analysis of a levan produced by Streptococcus salivarius SS2. Carbohydr. Res. 208: 193- 198.
  21. Stark, Y. and J. Leibovici. 1986. Different effects of the polysaccharide levan on the oncogenicity of cells of two variants of Lewis lung carcinoma. Br. J. Exp. Pathol. 67: 141-147.
  22. Whiting, G. and R. Coggins. 1967. Levan formation of Acetomonas. J. Inst. Brew. 73: 422- 425.
  23. Yamada, H. 1996. Contribution of pectins on health care, pp. 173- 190. In J. Visser and A. G. J. Voragen (eds.). Pectins and Pectinases.