Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Analysis of N- Terminal Amino Acid Sequence of Catechol 2,3-dioxygenase from Aniline Degrading Delftia sp. JK-2

Aniline 분해세균 Delftia sp. JK-2에서 분리된 Catechol 2,3-dioxygenase의 N-말단 아미노산 서열 분석

  • Hwang Seon-Young (Department of Life Science, Soonchunghyang University) ;
  • Kahng Hyung-Yeel (Department of Environmental Education, Sunchon National University) ;
  • Oh Kye-Heon (Department of Life Science, Soonchunghyang University)
  • 황선영 (순천향대학교 자연과학대학 생명과학부) ;
  • 강형일 (순천대학교 환경교육과) ;
  • 오계헌 (순천향대학교 자연과학대학 생명과학부)
  • Published : 2005.03.01
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Abstract

The aim of this work was to investigate the N-terminal amino acid sequence of catechol 2,3-dioxygenase isolated from Delftia sp. JK-2, which could utilize aniline as sole carbon, nitrogen and energy source. Molecular weight of the enzyme was determined to approximately 35 kDa by SDS-PAGE. N-terminal amino acid sequence of C2,3O from strain JK-2 was $^1MGVMRIGHASLKVMDMDAAVRHYENV^{26}$, and exhibited high sequence similarity with that of C2,3O from Pseudomonas sp., Comamonas sp. JS765, Comamonas test-osteroni, or Burkholderia sp. RP007. Approximately 950-bp C2,3O was obtained through PCR using the primers derived from N-terminal amino acid sequence. Analysis of the DNA sequence revealed that the deduced 296 amino acid sequences were determined, and it showed $100\%$ identity with C2,3O from Pseudomonas sp. AW-2 and $97\%$ similarity with Comamonas sp. JS765.

본 연구에서는 이 전 연구에서 단일 탄소원과 질소원 및 에너지원으로 aniline을 이용하는 Delftia sp. JK-2에서 분리, 정제된 바 있는 C2,3O의 N-말단 아미노산과 DNA 서열을 분석하였다. Aniline에서 배양한 Delftia sp. JK-2에서 분리된 약 35kDa의 C2,3O의 N-말단 아미노산 서열을 분석 한 결과 $^1MGVMRIGHASLKVMDMDAAVRHYENV^{26}$로 Pseudomonas sp. AW-2와 Comamonas sp. JS765의 C2,3O와 일치하는 것으로 나타났다. 위에서 확인된 아미노산 서열을 바탕으로 제작된 primer와 JK-2의 total genomic DNA를 기질로 사용하여 PCR을 수행한 결과 약 950 bp의 유전자 증폭산물을 획득하였다. 이 증폭산물 중 정확히 확인된 890 bp의 염기서열을 분석한 결과 Delftia JK-2의 C2,3O유전자 염기서열은 Pseudomonas su. AW-2의 C2,3O와 일치하였으며 Comamonas sp. Js765의 C2,3O와 $97\%$의 높은 상동성을 나타내었다.

Keywords

References

  1. Aoki, K., Y. Nakanishi, S. Murakami, and R. Shinke. 1990. Microbial metabolism of aniline through a meta-cleavage pathway: isolation of strains and production of catechol 2,3-dioxygenase. Agric. Biol. Chem 54, 205-206 https://doi.org/10.1271/bbb1961.54.205
  2. Bollag, D.M., M. D. Rozycki, and S. J. Edelstein. 1996. Gel electrophoresis under denaturing condition. New York, NY, USA. 2nd ed, 107-2
  3. Bugg, T.D.H. 2001. Oxygenases: mechanisms and structural motifs for $O_2$ activation. Curr. Opin. Chem. Biol. 5, 550-555 https://doi.org/10.1016/S1367-5931(00)00236-2
  4. Cho, Y.S., H.Y. Kahng, H.W. Chang, and K.H. Oh. 2000. Characterization of aniline-degrading bacterium, Delftia sp. JK-2 isolated from activated sludge of municipal sewage treatment plant. Kor. J. Microbiol. 36, 79-83
  5. Duffner, F.M., U. Kirchner, M.P. Bauer, and R. Muller. 2000. Phenol/ cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway. Gene 256, 215-221 https://doi.org/10.1016/S0378-1119(00)00352-8
  6. Hwang, S.Y., J.W. Chun, and K.H. Oh. 2004. Characterization of different dioxygenases isolated from Delftia sp. JK-2 capable of degrading aromatic compounds, aniline, benzoate, and p-hydroxybenzoate. Kor. J. Biotechnol. Bioeng. 19, 50-56
  7. Konopka, A., D. Knight, and R.F. Turco. 1989. Characterization of a Pseudomonas sp. capable of aniline degradation in the presence of secondary carbon sources. Appl. Environ. Microbiol. 55, 385-389
  8. Liu, Z., H. Yang, Z. Huang, P. Zhou, and S.J. Liu. 2002. Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3. Appl. Microbiol. Biotechnol. 58, 679-682 https://doi.org/10.1007/s00253-002-0933-8
  9. Milo, R.E., F.M. Duffner, and R. Muller. 1999. Catechol 2,3-dioxygenase from the thermophilic, phenol-degrading Bacillus thermoleovorans strain A2 has unexpected low thermal stability. Extremophiles 3, 185-190 https://doi.org/10.1007/s007920050115
  10. Murakami, S., Y. Nakanishi, N. Kodama, S. Takenaka, R. Shinke, and K. Aoki. 1998. Purification, characterization, and gene analysis of catechol 2,3-dioxygenase from the aniline-assimilating bacterium Pseudomonas species AW-2. Biosci. Biotechnol. Biochem.62, 747-752 https://doi.org/10.1271/bbb.62.747
  11. Ornston, L.N. 1966. The conversion of catechol and protocatechuate to $\beta$-ketoadipate by Pseudomonas putida. J. Biol. Chem. 241, 3776-3786
  12.  Parales, R.E., T.A. Ontl, and D.T. Gibson. 1997. Cloning and sequence analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain Comamonas sp. JS765. J. Ind. Microbiol. Biotechnol. 19, 385-391 https://doi.org/10.1038/sj.jim.2900420
  13.  Provident, M.A., J. Mampel, S. Macsween, A.M. Cook, and R.C. Wyndham. 2001. Comamonas testosteroni BR 6020 possesses a single genetic locus for extradiol cleavage of protocatechuate. Microbiology 147, 2157-2167 https://doi.org/10.1099/00221287-147-8-2157
  14.  Reineke, W., and H.J. Knacmuss. 1998. Microbial degradation of haloaromatic. Annu. Rev. Microbiol. 42, 263-287 https://doi.org/10.1146/annurev.mi.42.100188.001403
  15.  Schlomann, M. 1994. Evolution of chlorocatechol catabolic pathway. Biodegradation 255, 735-752
  16.  Schreiner, A., K. Fuchs, F. Lottspeich, H. Poth, and F. Lingens. 1991. Degradation of 2-methylaniline in Rhodococcus rhodochrous: cloning and expression of two clustered catechol 2,3-dioxygenase genes from strain CTM. J. Gen. Microbiol. 137, 2041-2048 https://doi.org/10.1099/00221287-137-8-2041
  17.  Yoko, N., S. Murakami, R. Shinke, and K. Aoki. 1991. Induction, purification, and characterization of catechol 2,3-dioxygenase from aniline-assimilating Pseudomonas sp. FK-8-2. Agric. Biol. Chem. 55, 1281-1289 https://doi.org/10.1271/bbb1961.55.1281