High-Level Production of Low-Branched Levan from Pseudomonas aurantiaca S-4380 for the Production of $di-\beta-D-Fructofuranose$ Dianhydride IV

  • JANG KI-HYO (Department of Food and Nutrition, Samcheok National University) ;
  • JANG EUN-KYUNG (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • KIM SEUNG-HWAN (Korea Food and Drug Administration) ;
  • KIM IN-HWAN (RealBioTech Co. Ltd.) ;
  • KANG SOON AH (Department of Fermented Food Science, Seoul University of Venture & Information) ;
  • KOH ISSAC (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology) ;
  • PARK YOUNG-IL (Division of Biotechnology, The Catholic University of Korea) ;
  • KIM YOUNG-JUN (Division of Biotechnology, The Catholic University of Korea) ;
  • HA SANG-DO (Department of Food Science and Technology, Chung-Ang University) ;
  • KIM CHUL HO (Laboratory of Metabolic Engineering, Korea Research Institute of Bioscience and Biotechnology)
  • Published : 2006.01.01

Abstract

The IscA gene, encoding a levansucrase of 424 amino acids (aa) residues, was cloned from the genomic DNA of Pseudomonas aurantiaca S-4380, and overexpressed in Escherichia coli. The recombinant levansucrase overexpressed in E. coli was then used to produce levan from sucrose. Levan crystals with 98% purity could be obtained from the reaction mixture with $62\%$ yield using an alcohol precipitation method. The molecular weight of the levan was $7\times10^5$ daltons. Methylation studies showed that the levan was branched: main linkage C-2,6; branched linkage C-2,1; and degree of branching $6\%$. Three bacterial levans from different strains were incubated with levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032, which produced $di-\beta-D-fructofuranose$ dianhydride IV (DFA IV); final conversion yields from the levans to DFA IV were $39\%$ in Zymomonas mobilis, $53\%$ in Serratia levanicum, and $59\%$ in P. aurantiaca S-4380 levansucrase. The levan from P. aurantiaca S-4380 levansucrase gave the highest conversion yield of levan to DFAIV so far reported.

Keywords

References

  1. Baik, B. H., Y. W. Lee, and Y. B. Lee. 1997. Antianemic agent containing iron and difructose. US patent 5,700,832
  2. Bradford, M. M. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248- 254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Defaye, J., A. Gadelle, and C. Pedersen. 1985. The behaviour of D-fructose and inulin towards anhydrous hydrogen fluoride. Carbohydr. Res. 136: 53-65 https://doi.org/10.1016/0008-6215(85)85185-5
  4. Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Department of Genetics, University of Washington, Seattle, U.S.A
  5. Han, Y. W. and M. A. Clarke. 1990. Production and characterization of microbial levan. J. Agric. Food Chem. 38: 393-396 https://doi.org/10.1021/jf00092a011
  6. Jang, E. K., K. H. Jang, I. Koh, I. W. Kim, S. H. Kim, S. A. Kang, C. H. Kim, S. D. Ha, and S. K. Rhee. 2002. Molecular characterization of the levansucrase gene from Pseudomonas aurantiaca S-4380 and its expression in Escherichia coli. J. Microbiol. Biotechnol. 12: 603-609
  7. Jang, K. H., K. B. Song, C. H. Kim, B. H. Chung, S. A. Kang, U. H. Chun, R. W. Choue, and S. K. Rhee. 2001. Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis. Biotechnol. Lett. 23: 339-344 https://doi.org/10.1023/A:1005641220946
  8. Kang, S. A., K. Hong, K. H. Jang, S. Kim, K. H. Lee, B. I. Chang, C. H. Kim, and R. Choue. 2004. Anti-obesity and hypolipidemic effects of dietary levan in high fat diet-induced obese rats. J. Microbiol. Biotechnol. 14: 796-804
  9. Kang, S. A., J. C. Lee, Y. M. Park, C. Lee, S. H. Kim, B. I. Chang, C. H. Kim, J. W. Seo, S. K. Rhee, S. J. Jung, S. M. Kim, and K. H. Jang. 2004. Secretory production of Rahnella aquatilis ATCC 33071 levansucrase expressed in Escherichia coli. J. Microbiol. Biotechnol. 14: 1232-1238
  10. Kim, C. H., E. K. Jang, S. H. Kim, K. H. Jang, S. A. Kang, K. B. Song, O. S. Kwon, and S. K. Rhee. 2005. Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV. Lett. Appl. Microbiol. 40: 228-234 https://doi.org/10.1111/j.1472-765X.2005.01658.x
  11. Kim, D. M. and D. W. Kim. 1999. Facile purification and characterization of dextransucrase from Leuconostoc mesenteroides B-512FMCM. J. Microbiol. Biotechnol. 9: 219-222
  12. Kim, H. J., H. E. Park, M. J. Kim, H. G. Lee, J. Y. Yang, and J. H. Cha. 2003. Enzymatic characterization of a recombinant levansucrase from Rahnella aquatilis ATCC 15552. J. Microbiol. Biotechnol. 13: 230-235 https://doi.org/10.1159/000075836
  13. Kim, M. G., C. H. Kim, J. S. Lee, K. B. Song, and S. K. Rhee. 2000. Synthesis of methyl ${\beta}$-D-fructoside catalyzed by levansucrase from Rahnella aquatilis. Enzyme Microbiol. Technol. 27: 646-651 https://doi.org/10.1016/S0141-0229(00)00267-2
  14. Kim, M. J., H. E. Park, H. K. Sung, T. H. Park, and J. Cha. 2005. Action mechanism of transfructosylation catalyzed by Microbacterium laevaniformans levansucrase. J. Microbiol. Biotechnol. 15: 99-104 https://doi.org/10.1159/000083642
  15. Kojima, I., T. Saito, M. Iizuka, N. Minamiura, and A. Ono. 1993. Characterization of levan produced by Serratia sp. J. Ferm. Bioeng. 75: 9-12 https://doi.org/10.1016/0922-338X(93)90169-9
  16. Lindberg, B., J. Lonngren, and J. L. Thompson. 1973. Methylation studies on levans. Acta Chem. Scand. 5: 1819- 1821
  17. Oh, I. K., S. H. Yoo, I. Y. Bae, J. Cha, and H. G. Lee. 2004. Effects of Microbacterium laevaniformans levans molecular weight on cytotoxicity. J. Microbiol. Biotechnol. 14: 985- 990
  18. Rochelle, P. A., J. C. Fry, R. J. Parkes, and A. Weightman. 1992. DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities. FEMS Microbiol. Lett. 100: 59-66 https://doi.org/10.1111/j.1574-6968.1992.tb05682.x
  19. Saito, K., H. Goto, and A. Yokota. 1997. Purification of levan fructotransferase gene from Arthrobacter nicotinovorans GS- 9 and production of DFA IV from levan by the enzyme. Biosci. Biotech. Biochem. 61: 1705-1709 https://doi.org/10.1271/bbb.61.1705
  20. Saito, K., T. Hira, T. Suzuki, H. Hara, A. Yokota, and F. Tomita. 1999. Effects of DFA IV in rats: Calcium absorption and metabolism of DFA IV by intestinal microorganisms. Biosci. Biotech. Biochem. 63: 655-661 https://doi.org/10.1271/bbb.63.655
  21. Saito, K. and F. Tomita. 2000. Difructose anhydrides: Their mass-production and physiological functions. Biosci. Biotechnol. Biochem. 64: 1321-1327 https://doi.org/10.1271/bbb.64.1321
  22. Saito, K., A. Yokoda, and F. Tomita. 1997. Molecular cloning of levan fructotransferase from Arthrobacter nicotinovorans GS-9 and its expression in Escherichia coli. Biosci. Biotech. Biochem. 61: 2076-2079 https://doi.org/10.1271/bbb.61.2076
  23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New York, U.S.A
  24. Sanger, F., S. Nichlen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467
  25. Song, K. B., K. S. Bae, Y. B. Lee, K. Y. Lee, and S. K. Rhee. 2000. Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan. Enzyme Microbiol. Technol. 27: 212-218 https://doi.org/10.1016/S0141-0229(00)00135-6
  26. Song, K. B., H. Belghith, and S. K. Rhee. 1996. Production of levan, a fructose polymer, using an overexpressed recombinant levansucrase. Ann. N. Y. Acad. Sci. 799: 601- 607 https://doi.org/10.1111/j.1749-6632.1996.tb33264.x
  27. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned gene. Meth. Enzymol. 185: 60-89 https://doi.org/10.1016/0076-6879(90)85008-C
  28. Tanaka, K., F. Yamaguchi, and S. Kusui. 1989. Action of levan fructotransferase of Arthrobacter ureafaciens on levan and phlein. Agric. Biol. Chem. 53: 1203-1211 https://doi.org/10.1271/bbb1961.53.1203