한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제20권5호
  • /
  • Pages.505-510
  • /
  • 1992
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  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

대장균에서 발현되는 Clostridium thermocellum의 섬유소 분해 효소의 특성

Properties of a Novel Clostridiclm thermocellum Endo-$\beta$-1,4-glucanase Expressed in Escherichia coli

  • 정경화 (한국과학기술원 생물공학과) ;
  • 이진호 (한국과학기술원 생물공학과) ;
  • 이용택 (한국과학기술원 생물공학과) ;
  • 김하근 (배재대학교 유전공학과) ;
  • 박무영 (한국과학기술원 생물공학과)
  • 발행 : 1992.10.01
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초록

고온성 혐기성 세균인 Clostridium thermocellum의 섬유소 분해 효소 유전자를 pUC9 플라스미드를 이용하여 대장균에 클로닝하였고, 지금까지 클로닝 된 C.thermocellum의 섬유소 분해 유전자들과 제한효소 양상을 비교하여 새로운 유전자임을 알 수 있었다. 대장균에서 섬유소 분해 효소를 열처리와 column chromatography에 의해서 정제를 하였고, 분자량은 40, 000이었다. 이 효소는 pH 5.0과 $65^{\circ}C$에서 CMC에 대해서 최대 활성을 보였고 최종 산물인 포도당과 cellobiose에 의한 활성의 저해는 크게 나타나지 않았다. CMC에 대한 이 효소의 $K_{m}$ 과 $V_{max}$값은 각각 0.39(w/v)와 268 U/mg protein이었다.

An endo-$\beta$-1,4-glucanase gene of Clostridium thermocellum was cloned in Escherichia coli and was considered as a novel gene by comparison with the restriction patterns of the C. thermocellum cellulase genes so far reported. The endoglucanase from recombinant E. coli was purified by column chromatography after heat treatment. The purified enzyme was a monomer having molecular weight of 40,000. The enzyme hydrolyzed CMC to glucose and cello-oligosaccharides at :naximum activities at pH 5.0 and $65^{\circ}C$. One of the endproducts, glucose, showed no inhibitory effect on the enzyme activity, while the other endproduct, cellobiose, inhibited slightly. The values of $K_{m}$ and $V_{max}$ of the enzyme for CMC were 0.39% (w/v) and 268 Ulmg protein, respectively.

키워드

참고문헌

  1. Enzyme Microb. Technol. v.3 Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products Bisaria,V.S.;T.K.Ghose
  2. Biotechnol. Bioeng. Symp. v.13 The cellusome-discret cell surface organelle of Clostridium thermocellum which exhibits separate antigenic, cellulose-binding and various cellulolytic activities Lamed,R.;E.Kenig;E.A.Bayer
  3. J. Bacteriol. v.162 Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum Beguin,P.;P.Cornet;J.P.Aubert
  4. Nucleic Acids Res. v.14 Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B Grepinet,O.;P.Beguin
  5. Gene v.63 Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridiuim thermocellum Schwarz,W.H.;S.Schimming;K.P.Rucknagel;S.Burgschwaiger;C.kreil;W.L.Staudenbauer
  6. Nucleic Acids Res. v.14 Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum Jollif,G.;P.Beguin;J.P.Aubert
  7. Gene v.69 Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for activity Hall,J.;G.P.Hazlewood;P.J.Barker;H.J.Gilbert
  8. Gene v.89 Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum Yague,E.;P.Beguin;J.P.Aubert
  9. Appl. Environ. Microbiol. v.51 Properties of a Clostridium thermocellum endoglucanase produced in Escherichia coli Schwarz,W.H.;F.Gr.Bnitz;W.L.Staudenbauer
  10. Biochimie. v.65 Identification of the endoglucanase encoded by the celB gene of Clostridium thermocellum Beguin,P.;P.Cornet;J.Millet
  11. Biochimie. v.68 Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli Petre,D.;J.Millet;R.Longin;P.Beguin;H.Girard;J.P.Aubert
  12. Bio/Technology v.4 Isolation, Crystallization and properties of a new cellulase of Clostridium thermocellum overproduced in Escherichia coli Joliff,G.;P.\B\'{e}guin;M.Juy;J.Millet;A.Ryter;Poljak,R.;J.-P.Aubert
  13. Appl. Environ. Microbiol. v.33 Fermentation of cellulose by Clostridium thermocellum in the absence and presence of Methanobacterium thermoautotrophicum Weimer,P.J.;J.G.Zeikus
  14. Molecular Cloning: A Laboratory Manual(2nd ed.) Sambrook,J.;E.F.Fritsch;T.Maniatis
  15. Enzyme Microb. Technol. v.8 Cloning and expression of a Bacillus cellulase gene in Escherichia coli Park,S.H.;M.Y.Pack
  16. Anal. Biochem. v.2 Measurement of carboxymethylcellulose activity Miller,G.L.;R.Blum;W.E.Glennon;A.L.Burton
  17. J. Biol. Chem. v.193 The protein measurement with folin phenol method Lowry,O.H.;N.J.Rosebrough;A.L.Farr;R.J.Randall
  18. Nature v.227 Clevage of structural proteins during the asembly of the head of bacteriophage T4 Laemmli,U.K.
  19. FEMS Microbiol. Lett. v.29 Cloning of ten distinct DNA fragments of Clostridium thermocellum coding for cellulases Millet,J.;D.Petre;P.Beguin;O.Raynaud;J.P.Aubert
  20. Biotechnol. Lett. v.7 Molecular cloning of Clostridium thermocellum genes involved in β-glucan degradation in bacteriophage lamda Schwarz,W.;K.Bronnenmeier;W.L.Staudenbauer
  21. J. Gen. Microbiol. v.133 Molecular cloning of Clostridium the rmocellum DNA and the expression of further novel endo-β-1,4-glucanase genes in Eschechia coli Romaniec,M.P.M.;N.G.Clarke;G.P.Hazlewood
  22. Appl. Microbiol. Biotechnol. v.27 High-level expression of Clostridium thermocellum cellulase genes in Escherichia coli Schwarz,W.H.;S.Schimming;W.L.Staudenbauer
  23. J. Biol. Chem. v.16 Mode of action and substrate specificities of cellulases from cloned bacterial genes Gilkes,N.R.;M.L.Langsford;D.G.Kilburn;R.C.Miller,Jr.;R.A.J.Warren