Development of Fusant Degrading Aniline and 4-chlorobiphenyl by Spheroplast Fusion between Pseudomonas sp. and Flavimonas oryzihabitans

Flavimonas oryzihabitans와 Pseudomonas sp.간 원형질체 융합에 의한 Aniline과 4-chlorobiphenyl 분해균주 개발

  • 박형수 (삼성엔지니어링 기술연구소) ;
  • 박용근 (고려대학교 생명공학원) ;
  • 김무훈 (삼성엔지니어링 기술연구소) ;
  • 고범준 (삼성엔지니어링 기술연구소) ;
  • 조미영 (삼성엔지니어링 기술연구소) ;
  • 김치경 (충북대학교 자연과학대학 미생물학과)
  • Published : 2000.12.01

Abstract

Spheroplast cell fusions were performed with Flavimonas oryzihabitans degrading aniline and Pseudomonas sp. degrading 4-chlorobiphenyl to develope the new fusant degrading aniline and 4-CBP and its characters were investigated. F. oryzihabitans was induced to antibiotic marker ($Cm^r$ by NTG treatment for the fusants selection. The results of spheroplast formation and regeneration frequencies of the strains treated with lysozyme-EDTA were 99% and 5.0~6.6%, respectively. Fusion products were treated with 40% (v/v) PEG 6000 and fusion frequency was $3.16{\times}10^{-4} $. The DNA content of fusant, F22 was approximately 2-fold compared with parents. The fusant was stable, and showed the mixed biochemical characteristics of the parent strains. F22 was similar to parent for cell growth pattern and degrading capacity on 5 mM aniline but cell growth rate of F22 was 1.5-fold higher than that of the parent on 10mM aniline. However 4-CBP degrading ability of F22 was slightly lower than that of parental strain.

아닐린분해능이 있는 Flavimonas oryzihabitans과 4-chlorobiphenyl (4-CBP)를 분해하는 Pseudomonas sp.를 원형질체 융합하여 아닐린과 4-CBP를 모두 분해하는 융합균주를 개발하고, 융합체의 특성을 조사하였다. F. oryzibhavitans는 융합체 선별표지 위애 NTG처리하여 chloramphenicol 내성 ($Cm^r$)을 유발하였다. Lysozyme-EDTA에 의한 각 모균주의 원형질체 생성율은 약 99%이었고, 원형질체의 재생율은 5.0~6.6%이었다. 원형질체는 40% PEG 6000을 사용하였을 때 효과적으로 융합이 이루어졌으며 융합율은 $3.16{\times}10^{-4} $이었다. 융합균인 F22의 DNA양는 모균주에 비해 약 2배 정도 증가 하였으며, 생화학적 특성은 모균주의 특성이 혼합하여 나타났다. F22는 5 mM 아닐린 최소배지에서는 모균주와 성장능과 분해능이 거의 비슷하였고, 10 mM 아닐린 최소배지에서는 1.5배 빠른 성장속도를 나타내었으나, 4-CBP 분해능은 모균주보다 약간 낮게 나타났다.

Keywords

References

  1. Science v.211 Biodegradation of chemicals environmental concern Alexander, M.
  2. J. Agric. Food Chem. v.16 Biochemical transformations of aniline herbicide in soil Bartha, R.
  3. Appl. Environ. Microbiol. v.53 Extensive degradation of Aroclors and environmentally transformed polychlorinated biphenyls by Alcaligenes eutrophus H850 Bedard, K. D.;R. E. Wagner;M. J. Brennon;M. L. Habert;J. F. Brown, Jr.
  4. Methods in Enzymology v.12 Determination of DNA concentration with diphenylamine Burton, K.;Colowick, S.P.(ed.);N. D. Kaplan(ed.)
  5. J. Bacteriol v.146 Plasmid specifying total degradation of 3-chlorbenzoate by a modified ortho pathway Chatterja, D.K.;S.T. Kellof;S. Hamada;A.M. Chakrabarty
  6. Appl. Environ. Microbiol. v.52 Protoplast formation and regeneration of dehydrodivanillin-degrading strains Fusobacterium varium and Enterococcus faecium Chen, W.;K. Ohmiya;S. Shimizu
  7. Appl. Environ. Microbiol. v.53 Intergeneric proplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanilline degradation Chen, W.;K. Ohmiya;S. Shimizu
  8. Master Thesis, Korea University Isolation and characterization of Flavimonas oryzihabitans KC-23 degrading aniline and restriction mapping of plasmid pKC-23 Choi, S.J.
  9. J. Gen. Microbiol. v.114 Genetic recombination in fused spheroplasts of Providence alcalifaciens Coetzee, J.N.;F.A. Sirqel;Lecatsas
  10. Appl. Environ. Microbiol. v.51 Construction of lactose-assimilating and high ethanol-producing yeasts by protoplast fusion Farahnak, F.;T. Seki;D. Y. Ryu;D. O. grydziak
  11. Proc. Natl. Acad. Sci., USA v.73 Fusion of protoplasts of Bacillus megaterium Fodor, K.;I. Alfoldi
  12. Microbiol. Rev. v.55 Microbiological degradation of pesticides in yard waste composting Fogarty, A. M.;O. H. Tuovinen
  13. J. Bacteriol. v.137 Electron microscopic study of Bacillus subtilis protoplast fusion Frehel, C.;A.M. Lheritire;C. Sanchez-Rivas;P. Scheffer
  14. J. Bacteriol v.137 Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts Gabor, M.H.;Hotchkiss, R.D.
  15. Hazardous wastes and Hazardous Materials v.3 no.4 Biodegradation of hazardous waste by conventional biological treatment Gardy. C.D.L.
  16. Kor. J. Microbiol. v.31 Cloning and experession of pcbAB genes from Pseudomonas sp. DJ-12 in Escherichia coli Han,J.J;T.K Sung;C.K. Kim
  17. Kor. J. Microbiol. v.30 Improvement of the strains degrading recalcitrant aromatic compounds by cell fusion between Arthrobacter spp. and Pseudomonas putida Hong,J.P;J.S. Lee;Y.N. Lee
  18. Ann. Rev. Microbiol. v.35 Genetic studies with bacterial protoplasts Hopweed, D.A.
  19. J. Gen. Microbiol. v.135 Protoplast isolation and regeneration in Streptomyces peptomyces calavuligerus Illing, G.T.;I.D. Normansell;J.F. Peberdy
  20. Kor. J. Microbiol. v.25 Isolation and characterization of bacteria degrading chlorinated aromatic hydrocarbons Kim, C.K.;J.W. Kim;Y.C. Kim
  21. Kor. J. Microbiol. v.30 Isolation and characterization of aniline-degrading bacteria Kahng, H.Y.;S.I. Kim;M.J. Woo;Y.K. Park;Y.N. Lee
  22. FEMS Microbiol. Lett. v.190 Characterization of strain HY99, a novel microorganism capable of aerobic and anaerobic degradation of aniline Kahng, H.Y.;J.J. Kukor;K.H. Oh
  23. Appl. Environ. Microbiol. v.43 Rapid screen for bacteria degrading water-insoluble, solid hydrocarbons on the agar plate Kiyohara, H.;K. Nagao;K. Yana
  24. Appl. Envioron. Microbiol. v.55 Characterization of Pseudomonas sp. capable of aniline degradation in the presence of secondary carbon sources Konopka, A.;D. Knight;R.F. Turco
  25. Kor. J. Microbiol. v.25 Properties of biparental clones formed by spheroplast fusion of Pseudomonas putida Lee, J.S.;Y.W. Lee;Y.N. Lee
  26. Kor. J. Microbiol. v.26 Spheroplast fusion of Pseudomonas sp. using plasmid as selection marker Lee, J.S.;M.R. Lee;Y.N. Lee
  27. Kor. J. Microbiol. v.31 Spheroplast Formation, Regeneration and Fusion of Flavimonas oryzihabitans KU21 Lee, S.Y.;Y.B. Lim;Y.K. Park;Y.N. Lee
  28. Kor. J. Microbiol. v.32 Improvement of the strains degrading aniline by spheroplast between Flavimonas oryzihabitans and Alcaligenes sp. Lee, S.Y.;J.S. Lee;Y.K. Park;Y.N. Lee
  29. Experiments in Molecular Genetics Miller, J.M.
  30. J. Bacteriol. v.139 High-frequency fusion of Streptomyces parvulus and or Streptomyces antibioticus protoplasts induced by polyethylene glycol Ochi, K.;M.J.M. Hitchcock;Kalz
  31. J. Gen. Microbiol. v.80 Formation and reversion of Streptomyces protoplasts: cultural condition and morphological study Okanishi, M., Suzuki;H. Wumezawa
  32. Bergey's manual of systematic bacteriology v.1 Palleroni, N.S.
  33. Kor. J. Microbiol. v.23 Electron microscopic obervations on protoplast fusion of Coryneform Bacteria Park, C.;B.S. Lim;M.J. Chun;W.K. Kim
  34. Residue Rev. v.76 Environmental and metabolic transformation of primary aromatic amines and related compounds Parris, G.E.
  35. Ann. Rev. Microbiol. v.33 Fungal protoplasts: Isolation, reversion and fusion Peberdy, J.F.
  36. Proc. Natl. Acad. Sci. USA. v.73 Fusion of bacteriol protoplasts Schaeffer, P.;B. Cami;R. Hotchkiss
  37. The Procaryotes Starr, M.P.;H. Stolop;H.G. Truper;A. Balows;H.G. Schlegel
  38. Methods in cell biology v.12 Analytical methods for yeast Stewart, P.R.;Prescott, D.M.(ed)
  39. Kor. J. Appl. Microbiol. Biotechnol. v.21 Enzymatic properties of the 2,3-dihydroxybiphenyl dioxygenase purified from Pseudomonas sp. DJ-12 Sung, T.K.;J.H. Nam;C.K. Kim
  40. Mikrobiologiya v.49 Aniline as the sole source of carbon, nitrogen, and energy for Alcaligenes faecalis Surortsera, E. G.;A. I. Bol'nova
  41. J. Bacteriol. v.83 Stabilization of protoplasts and spheroplasts by spermine and other polyamines Tabor, C.W.
  42. Bull. Environ. Contam. Toxicol. v.14 Activated sludge primary biodegradation of polychlorinated biphenyls Tucker, E.S.;V.W. Saeger;O. Hicks
  43. Appl. Environ. Microbiol. v.56 Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments Walia, S.;A. Khan;N. Rosenthal
  44. Appl. Environ. Microbiol. v.51 Evolved aniline actabolism in Acinetobacter calcoaceticus during continuous culture of river water Wyndham, R.C.