Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Sequence Analysis of a Levansucrase Gene from Rahnella aquatilis ATCC15552

  • Kim, Hyun-Jin (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Yang, Ji-Young (Division of Food Science nd Biotechnology, Pukyung National University) ;
  • Lee, Hyeon-Gye (Department of Food and Nutrition, Hanyang University) ;
  • Cha, Jae-Ho (Division of Biological Sciences, College of Natural Sciences, Pusan National University)
  • Published : 2001.08.01
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Abstract

An intracellular levansucrase gene, lscR from Rahnella aquatilis ATCC 15552, was cloned and its nucleotide sequence was determined. Nucleotide sequence analysis of this gene revealed a 1,238 bp open reading frame coding for a protein of 415 amino acids. The levansucrase was expressed by using a T7 promoter in Escherichia coli BL21 (DE3) and the enzyme activity was detected in the cytoplasmic fraction. The optimum pH and temperature of this enzyme for levan formation was pH 6 and $30^{\circ}C$, respectively. The deduced amino acid sequence of the lscR gene showed a high sequence similarity (59-89%) with Gram-negative levansucrses, while the level of similarity with Gram-positive enzymes was less than 42%. Multiple alignments of levansucrase sequences reported from Gram-negative and Gram-positive bacteria revealed seven conserved regions. A comparison of the catalytic properties and deduced amino acid sequence of lscR with those of other bacterial levansucrases strongly suggest that Gram-negative and Gram-positive levansucrases have an overall different structure, but they have a similar structure at the active site.

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References

  1. Infect. Immun. v.58 Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingritt Banas, J.A.;R. R. Russell;J.J. Ferretti
  2. 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
  3. Ann. N. Y. Acad. Sci. v.750 Enzymatic synthesis of low-calorie sugar suhstitutes: Cellobiofructose and gentiobiofructose Biton, J.;J.M. Michel;D. Le Beller;V. Pelence;F. Paul;P.F. Monsan;G. Gellf
  4. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Bradford, M. M.
  5. Biochem. J. v.279 Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis Chambert, R.;M.-F. Petit-Glatron
  6. Structure v.3 Structures and mechanisms of glycosylhydrolases DAvies, G.;B. Henrissat
  7. Physiol. Mol. Plant. Pathol. v.42 Characterization and influence on virulence of levansucrase gene from the fireblight pathogen Erwinia amylovora Geier, G.;K. Geider
  8. Appl. Microbiol. v.35 Microbial Levan Han, Y.W.
  9. Appl. Environ. Microbiol. v.64 Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrease gene 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
  10. J. Microbiol. Biotechnol. v.10 Cloning and sequencing of the α-1→6 dextransucrase gene from Leuconostoc mesenteroides B-742CB Kim, H.;D. Kim;H. J. Ryu;J. F. Robyt
  11. Biosci. Biotechnol. Biochem. v.59 Cloning and characterization of Zymomonas mobilis genes encoding extracellular levansucrase and invertase Kyono, K.;H. Yanase;K. Tonomura;H. Kawasaki;T. Sakai
  12. Nature v.227 Cleavage of structural proteins during the assembly of the of bacteriophage T4 Laemmli, U. K.
  13. Anal. Chem. v.31 Use of dinitrosalicylic acid reagent for determination of reducing sugar Miller, G. L.
  14. FEBS Lett v.448 Conserved sequence motifs in levansucrases and bifunctional β-xylosidascs and α-L-arabinases Naumoff, D. G.
  15. Biotechnol. Lett. v.13 Identification of saccharolytic enzymes of Zymomonas mobilis CP4 O'Mullan, P. J.;M. Szakacs-Dobozi;D. E. Eveleigh
  16. Biosci biotech. Biochem. v.56 Characterization of levansucrase from Rahnella aquatilis JCM-1683 Ohtsuka, K.;S. Hino;T. Fukushima;O. Ozawa;T. Kanematsu;T. Uchida
  17. 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. I. Song;J. W. Kwak;S. B. Lee;Y.W. Nam;J. S. Shin;Y. I. Park;S.K. Rhee;K.H. Paek
  18. Eur. J. Biochem. v.163 Secretion of Bacillus subtilis levansucrase:A possible two-step mechanism Petite-Glatron M.-E.;E. Benyahia;R. Chambert
  19. J. Bacteriol. v.175 The cell-bound fructorsyl-transferase of Struptococcus salivarius: The carboxyl terminus specifies specifies attachment in a Streptococcus gordonii model system Rathsam, C. ;P. M. Giffard;N. A. Jacques
  20. Molecular Coning. A Laboratory Manual(2nd ed.) Sambrook, J.;E. F. Fritsch;T. Maniatis
  21. J. Biotechnol. v.81 Molecular cloning of a gene encoding the thermoactive levansucrase from Rahnella auatilis and its growth phase-dependent expression in Escherichia coli Seo, J.-W.;K.-B. Song;K.-H. Jang;C.-H. Kim;B.-H. Jung;S.-K. Rhee
  22. Biochim. biophys. Acta v.1173 Nucleotide sequence of levansuvrase gene (levU) of Zymomonas mobilis ZMI (ATCC10988) Song, K.-B.;H.-K. Joo;S.-K. Rhee
  23. Biotechnol. Lett. v.16 Enzymatic synthesis of levan by Zymomonas mobilis levansucrase overexpressed in Escherichia coli Song, K.-B.;S.-K. Rhee
  24. J. Mol. Biol. v.189 Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes Studier, F.W.;B.A. Moffatt
  25. Nucleic Acides Res. v.22 CKISTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice thompson, J.D.;D.G. Higgins;T.J. Gibson