생명과학회지 (Journal of Life Science)

  • 제7권4호
  • /
  • Pages.253-261
  • /
  • 1997
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지

E. coli에서 글루타치온 생산 증가를 위한 재조합 플라스미드의 구성

Construction Various Recombiant Plasmids for the Enhancement of Glutathione Production in E. coli.

  • 남용석 (고려대학교 법의학교실) ;
  • 이세영 (고려대학교 생명공학원)
  • 발행 : 1997.12.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

E. coli에서 글루타치온 생산 증가를 위해서 E. coli에서 분리한 gehI과 gshII유전자를 함유하고 있는 여러 재조합 플라스미드를 구성하여 도입하였다.pBR325 벡터에 gehI 유전자를 각각 1-3개를 포함한 재조합 플라스미드 및 gehI과 gehII 유전자를 동시에 갖는 재조합 플라스미드를 구성하였다. 계속적으로 반복된 gehI 유전자가 증폭된 E. colidml $\gamma $-gluramylcysteine synthetase의 효소활성은 삽입된 gehI 유전자의 부에 따라 증가하였다. 구성된 재조합 플라스미드를 함유한 E.coli의 글루타치온 생산능을 accetate kinase반응을 ATP재생계로 사용하여 조사한 결과 반복된 gehI 유전자를 함유한 E.coli의 글루타치온 생산능력은 삽입된 gehI 유전자의 수에 비례한여 증가하였으며, gehI 유전자의 추가적인 도입에 의해 글루타치온 생산능력은 2배 증가하였다. E.coli에서 글루타치온의 효소적 생산은 주로 \gamma $-gluramylcysteine synthetase의 효소활성에 의해 영향을 받았다. 가장 높은 글루타치온 생산능은 pGH501 (pUC8-gsh.I.II.III) 플라스미드를 갖는 균주에서 관찰되었다.

In order to enhance glutathione production, various recombinant plasmids containing gshI and/or gshII genes isolated from E. coli K-12 were constructed and introduced into E. coli. Some plasmids contained one to three copies of gshI genes in pBR325 and others contained both gshI and genes for glutathione biosynthesis. $\gamma$-Glutamylcysteine synthetase activities of E, coli strains amplified tandem repeated gshI genes were dependent on the number of inserted gshI genes. The glutathione productivity of E. coli strains harboring various plasmids was investigated using an E. coli acetate kinase reaction as an ATP regenerating system. The glutathione productivity of E. coli strains harboring tandem repeated gshI genes was increased in proportion to the number of inserted gshI genes. By the introduction of gshII gene, the glutathione productivity of the E. coli was increased by two-fold compared with E. coli strain amplified gshI gene only. The enzymatic production of glytathione in E. coli was mainly affected by the increase of $\gamma$-glutamylcysteine synthetase activity. The highest glutathione productivity was obtained in E. coli strains harboring pGH-501 plasmid containing two copies of gshI and copy of gshII genes in pUC8 vector.

키워드

참고문헌

  1. Glutathione. Ann. Rev. Biochem. v.57 Meister, A.;Anderson, M. E.
  2. Ann. Rev. Biochem. v.45 Glutathione and related γ-glutamyl compounds : Biosynthesis and utilization Meister, A.;Tate, S. S.
  3. Glutathione and r-glutamylpeptide. In Biotechnology of amino aced production v.24 Kumagai, H.
  4. Eur. J. Appl. Microbial. Biotechnol. v.6 Continuous production of glutathione by immobilized Saccharomyces cerevisiae cells Murata, K.;Tani, K.;Kato, J.;Chibata, I.
  5. J. Appl. Biochel. v.1 Application of immobilized ATP in the production of glutathione by a multienzyme system Murata, K.;Tani, K.;Kato, J.;Chibata, I.
  6. Biochimie v.62 Continuous production of glutathione using immobilized microbial cells containing ATP generating system Murata, K.;Tani, K.;Kato, J.;Chibata, I.
  7. Enz. Microb. Technol. v.3 Glycolytic pathway as an ATP generating system and its application to the production of glutathione and NADP Murata, K.;Tani, K.;Kato, J.;Chibata, I.
  8. Appl. Environ. Microbiol. v.44 Cloning of a gene responsible for the biosynthesis of glutathione in Escheichia coli B. Murata, K.;Kimura, A.
  9. Agri. Biol. Chem. v.47 Cloning and amplification of a gene for glutathione synthetase in Escherichia coli B. Murata, K.;Miya, T.;Gushima, H.;Kimura, A.
  10. Ph. D. dissertation Korea University Molecular breeding of glutathione bacterial strains Nam, Y. S.
  11. Kor. J. Appl. Microbial. Biothechnol v.19 no.6 Cloning of genes for the biosynthesis of glutathione from E. coli K-12 Nam, Y. S.;Park, T.-I.;Lee, S.-Y.
  12. J. Appl. Biochem. v.5 Construction of glutathione producing strains of Escherichia coli B by recombinant DNA techniques Gushima, H.;Miya, T.Murata, K.;Kimura, A.
  13. Gene v.8 Plasmids containing many copies of a synthetic lactose operator Sadler, J. R.;Tecklenberg, M.;Batz, J. L.
  14. J. Micro. Biotech. v.1 no.3 Amplification of glutathione production in E. coli cells using recombinant DNA techniques Nam, Y.-S.;Park, Y.-I.;Lee, S.-Y.
  15. A Laboratory Manual(2nd. ed) Molecular Cloning Sambrook, J.;Fritsh, E. F.;Maniatis, T.
  16. Biochem. J. v.111 Studies in the enzymology of glutathione metabolism in human erythrocytes Jackson, C. R.
  17. Bilchem. Pharmacol. v.15 Estimation and identification of thiols in rat spleen after cysteine of glutathione treatment : Relevance to protection against nitrogen mustards Ball, C. R.
  18. J. Biol. Chem. v.193 Protein measurement with the folin phenol reagent Lowry, O. J.;Resenbrough, N. J.;Farr, A. L.;Randall, R. J.
  19. Anal. Biochem. v.27 Enzymatic methods for quantitatine determination of nanogram amounts of total and oxidized glutathione : Application to mammalian blood and other tissue Tietze, F.
  20. J. Biol. Chem. v.254 Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose opreator and pomoter Hardies, S. C.;Patient, P. K.;Klein, R. D.;Reznicoff, W. C.;Wells, R. D.
  21. Nature v.302 Variable amplification of immunoglobulin light chain genes in human populations Taub, R. A.;Hollis, G. F. Hieter, P. A.;Korsmeyer, S.;Waldmann, T. A.;Leder, P.
  22. Eur. J. Appl. Microbial. Biotechnol v.10 Glutathione production coupled with an ATP regeneration system Murata, K.;Tani, K.;Kato, J.;Chibata, I.
  23. Eur. J. Appl. Microbial. Biltechnol. v.11 Glutathione production by immobilized Saccharomyces cerrvisiae cells containing an ATP generation system Murata, K.;Tani, K.;Kato, J.;Chibata, I.