Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning of Pseudomonas sp.Inulinase Gene and its Expresstion in E. coli

Pseudomonas sp. Inulinase 유전자의 클로닝 및 Escherichia coli에서의 발현

  • 엄수정 (고려대학교 자연자원대학 유전공학과) ;
  • 권영만 (고려대학교 자연자원대학 유전공학과) ;
  • 최용진 (고려대학교 자연자원대학 유전공학과)
  • Published : 1995.10.01
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Abstract

A strain of Pseudomonas sp. isolated from soil was shown to produce a high level of extracellular endo-inulinase. In this work, the endo-inulinase gene (inu1) of the bacterial strain was cloned into the plasmid pBR322 by using EcoRI restriction endonuclease and E. coli HB101 as a host strain. One out of 7, 000 transformants obtained from the above cloning experiment formed a clear zone around its colony on the selective medium supplemented with 2.0% inulin after a prolonged incubation at 37$\circ$C and subsequent cold shock treatment. The functional clone was found to carry a recombinant plasmid (pKMG50) with a 3.7 kb genomic insert containing the genetic information for the inulinase activity. The inulinase from E. coli HB101/pKMG50 was proved to be an endo-acting enzyme and produced constitutively in the recombinant E. coli cells. Zymogram of the enzyme from the recombinant cells with inulin substrate indicated that the molecular mass of the active protein was 190 Kd, while that of the endo-inulinase from the Pseudomonas strain was 170 Kd. This size discrepancy suggested that the inulinase from the recombinant E. coli HB101 cells might be the initial product of translation, not the mature form produced in the strain of Pseudomonas sp..

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References

  1. J. Chem. Soc, v.72 Studies on fructosans Hirst,E.L.;D.J.Mcgilvray;E.G.V.Percival
  2. Enzyme Microbiol. Technol. v.3 Enzymatic hydrolysis of plant extracts containing inulin Guriraud,J.P.
  3. Kor. J. Appl. Microbiol. Bioeng. v.15 Cluture condition for inulase production by Pseudomonas sp. Lee,T.K.;H.C.Sung;Y.J.Choi;H.C.Yang
  4. Kor. J. Appl. Microbiol. Bioeng. v.16 Purification and properties of extracelluar inulase of Pseudomonas sp. Lee,T.K.;Y.J.Choi;H.C.Yang
  5. Kor. J. Appl. Microbiol. Bioeng. v.16 Characteristics of extracelluar endo-inulinase produced by Pseudomonas sp. Lee,T.K.;H.C.Shin;Y.J.Choi;H.C.:Yang
  6. Complete nucleotied sequence of the E.coli plasmid pBR322 v.16 Sutcloffe,J.G.
  7. Molicular Cloning, A Laboratory Manual Maniatis,T.;E.F.Fritsch;J.Sambrook
  8. Recombinant DNA Techniques. An introduction Rodriguez,R.L.;R.C.Tait
  9. Techniques for Transformation of E.coli v.1 Hanahan,K.;D.M.Glover(ed.)
  10. J. Mol. Biol. v.98 Detection of specific sequences among DNA fragments separated by gel electrophoresis Soutern,E.
  11. Nucl. Acids. Res. v.13 Nonradioactive hybridization probe prepared by the chemical labeling of DNA and RNA with a novel reagent, photobiotin Anthony,C.F.;L.M.James
  12. Anal. Chem. v.31 Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar Miller,G.L.
  13. J. Biol. Chem. v.193 Protein measurement with Folin phenol reagent Lowry,O.H.;N.J.Rasebrough;A.L.Farr;R.J.Randall
  14. Nature v.227 Cleavage of structural proteins during the Assembly of the Head of Bacteriophage T4 Laemnli,U.K.
  15. Journal of Chromatography v.369 Determination of inulase isoenzymes on polyacrylamide gels by activity staining R.H.F.Beck;W.Praznik
  16. Biochem. v.27 The metabolism of fructose polymers in plant Edelman,J.;Dikerson,A.G.
  17. Anal. Chem. v.27 Determination of easily hydrolyzable fructose units on dextrin preparations Wise,C.S.;Dimler,R.J.;Davis,H.A.;Rist,C.E.