Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Characterization of the Levansucrase Gene from Pseudomonas aurantiaca S-4380 and Its Expression in Escherichia coli

  • Jang, Eun-Kyung (RealBioTech Co. Ltd., Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Jang, Ki-Hyo (Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Isaac Koh (Biotechnology Research Division, KRIBB) ;
  • Kim, In-Hwan (RealBioTech Co. Ltd., Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Kim, Seung-Hwan (RealBioTech Co. Ltd., Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Kang, Soon-Ah (Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University) ;
  • Kim, Chul-Ho (RealBioTech Co. Ltd., Korea Research Institute of Bioscience and Biotechnology(KRIBB), Biotechnology Research Division, KRIBB) ;
  • Ha, Sang-Do (Department of Industry and Technology, Korea Health Industry Development Institute) ;
  • Rhee, Sang-Ki (Biotechnology Research Division, KRIBB)
  • Published : 2002.08.01
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Abstract

DFA IV is di-D-fructose-2,6':6,2'-dianhydride, consisting of two fructose residues. It can be enzymatically synthesized from levan by levan fructotransferase, and can be used for mineral absorption. Understanding of the structure and composition of levan is important to obtain high-level production of DFA IV. A bacterial strain, Pseudomonas aurantiaca 5-4380, was identified to produce low-branched levan, and the levansucrase gene (lsch) from this bacterium was found to be composed of 1,275 Up coding for a protein of 424 amino acids, with an estimated molecular weight of 47 kDa. The bacterial levansucrase gene was expressed in Escherichia coli DH5${\alpha}$ by its own promoter and lac promoter. The recombinant levansucrase was produced in soluble form with 170U of levansucrase activity from 1-ml E. coii culture broth. The expressed enzyme from the clone showed similar biochemical properties, such as size of active levansucrase, degree of branching, and optimum temperature, with P.aurantiaca 5-4380 levansucrase.

Keywords

References

  1. Microbiology v.142 Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4 Arrieta, J.;L. Hernandez;A. Coego;V. Suarez;E. Balmori;C. Menendez;M. F. Petit-Glatron;R. Chambert;G. Selman-Housein https://doi.org/10.1099/13500872-142-5-1077
  2. US Patent 5,700,832 Antianemic agent containing iron and difructose Baik, B. H.;Y. W. Lee;Y. B. Lee
  3. Biochem. J. v.337 Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity Batista, F. R.;L. Hernandez;J. R. Fernandez;J. Arrieta;C. Menendez;R. Gomez;Y. Tambara;T. Pons https://doi.org/10.1042/0264-6021:3370503
  4. Anal. Biochem. v.72 A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding Bradford. M. M. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Eur. J. Biochem. v.41 Kinetic studies of levansucrase of Bacillus subtilis Chambert, R.;G. Treboul;R. Dedonder https://doi.org/10.1111/j.1432-1033.1974.tb03269.x
  6. J. Microbiol. Biotechnol. v.11 Levan-producing Bacillus subtilis BS 62 and its phylogeny based on its 16s rDNA sequence Choi, S. Ⅱ.;C. Sung;W. Y. Choi
  7. PHYLIP (Phylogeny Inference Package)(version 3.5c) Felsenstein, J.
  8. J. Bacteriol. v.153 Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: Expression of the gene in Escherichia coli Gay, P.;D. Le Coq;M. Steinmetz;E. Ferrari;J. A. Hoch
  9. Physiol. Mol. Plant. Pathol. v.42 Characterization and influence on virulence of levansucrase gene from the fireblight pathogen Erwinia amylovora Geier, G.;K. Geider https://doi.org/10.1006/pmpp.1993.1029
  10. J. Agric. Food Chem. v.38 Production and characterization of microbial levan Han, Y. W.;M. A. Clarke https://doi.org/10.1021/jf00092a011
  11. Appl. Environ. Microbiol. v.64 Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola Hettwer, U.;F. R. Jaeckel;J. Boch;M. Meyer;K. Rudolph;M. S. Ullrich
  12. J. Microbiol. Biotechnol. v.11 Cloning and sequence analysis of a levansucrase gene from Rahnella aquatilis ATCC15552 Kim, H. J.;J. Y. Yang;H. G. Lee;J. H. Cha
  13. J. Ferm. Bioeng. v.75 Characterization of levan produced by Serratia sp. Kojima, I.;T. Saito;M. Iizuka;N. Minamiura;A. Ono https://doi.org/10.1016/0922-338X(93)90169-9
  14. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli, U. K. https://doi.org/10.1038/227680a0
  15. Biochim. Biophys. Acta v.1353 A novel levansucrase-levanase gene cluster in Bacillus stearothermophilus ATCC 12980 Li, Y.;J. A. Triccas;T. Ferenci https://doi.org/10.1016/S0167-4781(97)00103-6
  16. J. Microbiol. Biotechnol. v.9 Transgenic tobacco plants expressing the bacterial levansucrase gene show enhanced tolerance to osmotic stress Park, J. M.;S. Y. Kwon;K. B. Song;J. W. Kwak;S. B. Lee;Y. W. Nam;J. S. Shin;Y. I. Park;S. K. Rhee;K. H. Paek
  17. FEMS Microbiol. Lett. v.100 DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities Rochelle, P. A.;J. C. Fry;R. J. Parkes;A. J. Weightman
  18. Biosci. Biotech. Biochem. v.61 Purification of levan fructotransferase gene from Arthrobacter nicotinovorans GS-9 and production of DFA Ⅳ from levan by the enzyme Saito, K.;H. Goto;A. Yokota;F. Tomita https://doi.org/10.1271/bbb.61.1705
  19. Biosci. Biotech. Biochem. v.63 Effects of DFA Ⅳ in rats: Calcium absorption and metabolism of DFA Ⅳ by intestinal microorganisms Saito, K.;T. Hira;T. Suzuki;H. Hara;A. Yokota;F. Tomita https://doi.org/10.1271/bbb.63.655
  20. Biosci. Biotechnol. Biochem. v.64 Difructose anhydrides: Their mass-production & physiological functions Saito, K.;F. Tomita https://doi.org/10.1271/bbb.64.1321
  21. Biosci. Biotech. Biochem. v.61 Molecular cloning of levan fructotransferase gene from Arthrobacter nicotinovorans GS-9 and its expression in Escherichia coli Saito, K.;A. Yokota;F. Tomita https://doi.org/10.1271/bbb.61.2076
  22. A Laboratory Manual(2nd ed.) Molecular Cloning Sambrook, J.;E. F. Fritsch;T. Maniatis
  23. Proc. Natl. Acad. Sci. USA v.74 DNA sequencing with chain-terminating inhibitors Sanger, F.;S. Nichlen;A. R. Coulson https://doi.org/10.1073/pnas.74.12.5463
  24. J. Biotechnol. v.81 Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella aquatilis and its growth phase-dependent expression in Escherchia coli Seo, J. W.;K. B. Song;K. H. Jang;C. H. Kim;B. H. Jung;S. K. Rhee https://doi.org/10.1016/S0168-1656(00)00281-9
  25. Enzyme. Microb. Technol. v.27 Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride Ⅳ formation from levan Song, K. B.;K. S. Bae;Y. B. Lee;K. Y. Lee;S. K. Rhee https://doi.org/10.1016/S0141-0229(00)00135-6
  26. Ann. N. Y. Acad. Sci. v.799 Production of levan, a fructose polymer, using an overexpressed recombinant levansucrase Song, K. B.;H. Belghith;S. K. Rhee https://doi.org/10.1111/j.1749-6632.1996.tb33264.x
  27. Biochem. J. v.344 Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975 Song, D. D.;N. A. Jacques https://doi.org/10.1042/0264-6021:3440259
  28. Biochim. Biophy. Acta v.1173 Nucleotide sequence of levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988) Song, K. B.;H. K. Joo;S. K. Rhee https://doi.org/10.1016/0167-4781(93)90130-6
  29. Biotechnol. Lett. v.16 Enzymatic synthesis of levan by Zymomonas mobilis levansucrase overexpressed in Escherichia coli Song, K. B.;S. K. Rhee
  30. Ann. N. Y. Acad. Sci. v.864 Levansucrase of Rahnella aquatilis ATCC33071. Gene cloning, expression, and levan formation Song, K. B.;J. W. Seo;M. G. Kim;S. K. Rhee https://doi.org/10.1111/j.1749-6632.1998.tb10369.x
  31. Thin-layer Chromatography: A Laboratory Handbook Stahl, E.
  32. DNA Res. v.7 Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005 Tajima, K.;T. Tanio;Y. Kobayashi;H. Kohno;M. Fujiwara;T. Shiba;T. Erata;M. Munekata;M. Takai https://doi.org/10.1093/dnares/7.4.237
  33. Gene v.96 Isolation and characterization of levansucrase-encoding gene from Bacillus amyloliquefaciens Tang, L. B.;R. Lenstra;T. V. Borchert;V. Nagarajan https://doi.org/10.1016/0378-1119(90)90345-R