Characterization of different Dioxygenases isolated from Delftia sp. JK-2 capable of degrading Aromatic Compounds, Aniline, Benzoate, and p-Hydroxybenzoate

방향족 화합물인 Aniline, benzoate, p-Hydroxybenzoate를 분해하는 Delftia sp. JK-2에서 분리된 Dioxygenases의 특성연구

  • 오계헌 (순천향대학교 생명과학부) ;
  • 황선영 (순천향대학교 생명과학부) ;
  • 천재우 (순천향대학교 생명과학부) ;
  • 강형일 (순천대학교 환경교육과)
  • Published : 2004.02.01

Abstract

The aim of this work was to investigate the purification and characterization dixoygenases isolated from Delftia sp. JK-2, which could utilize aniline, benzoate, and p-hydroxybenoate as sole carbon and energy source. Catechol 1,2-dioxygenase (C1, 2O), catechol 2,3-dioxygenase(C2, 3O), and protocatechuate 4,5-dioxygenase(4,5-PCD) were isolated by benzoate, aniline, and p-hydroxybenzoate. In initial experiments, several characteristics of C1 ,2O, C2, 3O, and 4,5-PCD separated with ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose were investigated. Specific activity of C1 ,2O, C2, 3O, and 4,5-PCD were approximately 3.3 unit/mg, 4.7 unit/mg, and 2.0 unit/mg. C1 ,2O and C2, 3O demonstrated their enzyme activities to other substrates, catechol and 4-methylcatechol. 4,5-PCD showed the specific activity to the only substrate, protocatechuate, but the substrates(e.g., catechol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol, 4-nitrocatechol) did not show any specific activities in this work. The optimum temperature of C1, 2O, C2, 3O, and 4,5-PCD were 30$^{\circ}C$, and the optimal pHs were approximately 8, 8, and 7, respectively. Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/ showed inhibitory effect on the activity of C1, 2O and C2, 3O, but Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/, Fe$\^$3+/ showed inhibitory effect on the activity of 4,5-PCD. Molecular weight of the C1, 2O, C2, 3O, and 4,5-PCD were determined to approximately 60 kDa,35 kDa, and 62 kDa by SDS-PAGE.

본 연구의 목적은 방향족 화한물인 aniline, benzoate, p-hydroxybenzoate를 분해할 수 있는 Delftia sp. JK-2에서 이들 각 기질에서 배양시 다른 종류의 dioxygenases를 분리 정제하고, 정제된 dioxygenases의 특성을 조사하기 위하여 실시하기 위한 것이다. 기질로서 benzoate, aniline, 또는 p-hydroxybenzoate에 따라 분리된 dioxygenases는 각각 catechol 1,2-dioxygenase (C1 ,2O), catechol 2,3-dioxygenase(C2, 3O), 그리고 protocatechuate 4,5-dioxygenase (4,5-PCD)였다. 각 dioxygenases의 특성을 조사하기 위하여 먼저 benzoate, aniline 또는 p-hydroxybenzoate에서 배양한 Delftia sp. JK-2 세포를 초음파 분쇄기로 파쇄하여, ammonium sulfate precipitation, DEAE-sepharose, 그리고 Q-sepharose의 순서로 정제하여 농축하였다. 정제$.$농축된 dioxygenases의 특이 활성도를 보면 C1, 2O는 3.3 unit/mg, C2, 3O는 4.7unit/mg이고, 4,5-PCD는 2.0 unit/mg이다 C1, 2O와 C2, 3O의 기질 특이성 조사에서는 catechol과 4-methylcatechol에서 두 효소 모두 효소 활성이 나타났으며, C1. 2O에서는 3-methylcatechol에서 약간의 활성이 확인되었고, 4,5-PCD는 protocatechuate에서만 효소 활성을 보여주었다. C1l, 2O와 C2, 3O는 3$0^{\circ}C$와 pH 8.0에서 최적의 활성을 나타내는 것으로 조사되었으며, 4,5-PCD는 3$0^{\circ}C$와 pH 7.0에서 최적의 활성이 조사되었다. Delftia sp. JK-2에서 정제된 C1, 2O와 C2, 3O의 효소활성은 Ag$^{+}$, Hg$^{+}$, 그리고 Cu$^{2+}$에 의해 억제되는 것으로 나타났으며, 4,5-PCD의 경우에는 Ag$^{+}$, Hg$^{+}$, 그리고 Cu$^{2+}$ 뿐만 아니라 Fe$^{3+}$ 에 이해서도 효소 활성이 억제되는 것이 확인되었다. C1, 2O, C2, 3O, 4,5-PCD의 분자량은 SDS-PAGE에 의해 각각 60kDa, 35kDa, 62kDa로 측정되었다.

Keywords

References

  1. Appl. Environ. Microbiol. v.48 Mechanisms and pathways of aniline elimination from aquatic environments Lyons,C.D.;S.Katz;R.Bartha
  2. Microbiology v.147 Comamonas testosteroni BR 6020 possesses a single genetic locus for extradiol cleavage of protocatechuate provident,M.A.;J.Mampel;S.Macsween;A.M.Cook;R.C.Wyndham https://doi.org/10.1099/00221287-147-8-2157
  3. Appl. Microbiol. Biotechnol. v.58 Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3 Liu,Z.;H.Yang;Z.Huang;P.Zhou;S.J.Liu https://doi.org/10.1007/s00253-002-0933-8
  4. J. Bacteriol. v.182 Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenases and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6 Masai,E.;K.Momose;H.Hara;S.Nishikawa;Y.Katayama;M.Fukuda https://doi.org/10.1128/JB.182.23.6651-6658.2000
  5. Kor. J. Microbiol. v.31 Purification and characterization of catechol 1,2-dioxygenase from aniline degrading Achromobacter gr. D.V.K-24 Kim,S.I.;S.H.Kim;Y.N.Lee
  6. Agric. Biol. Chem. v.48 Rapid biodegradation of aniline by Frateuria species ANA-18 and its aniline metabolism Aoki,K.;K.Ohtsuka;R.Shinke https://doi.org/10.1271/bbb1961.48.865
  7. Agric. Biol. Chem. v.54 microbial metabolism of aniline through a meta-cleavage pathway: Isolation of strains and production of catechol 2,3-dioxygenase Aoki,K.;Y.Nakanishi;S.Murakami;R.Shinke https://doi.org/10.1271/bbb1961.54.205
  8. J. Prot. Chem. v.19 Purification and catalytic properties of two catechol 1,2-dioxygenae isozyme from benzoate-grown cells of Acinetobacter radioresistens Briganti,F.;E.Pessione;C.giunta;A.Scozzafava https://doi.org/10.1023/A:1007116703991
  9. Eur. J. Biochem. v.229 Substrate specificity differences between two carechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida Cerdan,P.;M.Rekik;S.Harayama https://doi.org/10.1111/j.1432-1033.1995.tb20445.x
  10. J. Bacteriol. v.170 Nucleotide sequence and regulational analysis genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22 (pTDNI) Fukumori,F.;C.P.Saint
  11. J. Basic. Microbiol. v.38 Utilization of quinate and p-hydroxybenzoate by actinomycetes; key enzymes and taxonomic relevance Ground,E.;H.J.Kutzner https://doi.org/10.1002/(SICI)1521-4028(199809)38:4<241::AID-JOBM241>3.0.CO;2-S
  12. J. Biochem. v.117 Overexpression of Pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered Escherichia coli Kobayashi,T.;T.Ishida;K.Horiike;Y.Takahara;N.Numao;A.Nakazawa;T.Nakazawa;M.Nozaki https://doi.org/10.1093/oxfordjournals.jbchem.a124753
  13. J. Gen. Microbiol. v.137 Degradation of 2-methylaniline in Rhodococcus rhodochrous: cloning and expression of two clustered catechol 2,3-dioygenase enes from strain CTM Schreiner,A.;K.Fuchs;F.Lottspeich;H.Poth;F.Lingens https://doi.org/10.1099/00221287-137-8-2041
  14. Agric. Biol. Chem. v.55 Induction, purification, and characterization of catechol 2,3-dioxygenase from aniline-assimilating Pseudomonas sp. FK-8-2 YoKo,N.;S.Murakami;R.Shinke;K.Aoki https://doi.org/10.1271/bbb1961.55.1281
  15. Biochim. Biophys. Acta v.9 Degradation of trans-ferulic and p-coumaric acid by Acinetobabcter calcoaceticus DSM 586 Delneri,E.;G.Degrassi;R.Rizzo;C.V.Brushi
  16. Kor. J. microbiol. v.36 Chracterization of aniline-degrading bacterium, Delftia sp. JK-2 isolated from activated sludge of municipal sewage treatment plant Cho,Y.S.;H.Y.Kahng;H.W.Chang;K.H.Oh
  17. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the priniple of protein-dye binding Bradford,M.M. https://doi.org/10.1016/0003-2697(76)90527-3
  18. Gel Electrophoresis under Denaturing Condition (2nd ed.) Bollag,D.M.;M.D.Rozycki;S.J.Edelstein
  19. J. Bacteriol. v.127 Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties Patel,R.N.;C.T.Hou;A.Felix;M.O.Lillard
  20. J. Ind. microbiol. Biotechnol. v.19 Cloing and sequence and analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain Comamonas sp. JS765 Parales,R.E.;T.A.Ontl;D.T.Gibson https://doi.org/10.1038/sj.jim.2900420
  21. Arch. Biochem. Biophys v.332 Characterization of the gene encoding catechol 2,3-dioxygenase of Alcaligenes sp. KF711: overexpression, enzyme purification, and nucleotide sequencing Moon,J.;K.R.Min;C.K.Kim;K.H.Min;Y.Kim https://doi.org/10.1006/abbi.1996.0339
  22. J. Biol. Chem. v.258 EPR and mossbauer studies of protocatechuate 4,5-dioxygenase Arciero,D.A.;J.D.Lipscomb;B.H.Huynh;T.A.Kent;E.Munck
  23. Arch. Miribiol. v.161 Purification and properties of a homodimeric protocatechuate 4,5-dioxygenase from Rhizobium leguminosarum Chen,Y.P.;C.R.Lovell
  24. Extremophiles v.3 Catechol 2,3-dioxygenase from the thermophilic, phenol-degrading Bacillus thermoleovorans strain A2 has unexpected low thermal stability Milo,R.E.;F.M.Duffner;R.Muller https://doi.org/10.1007/s007920050115
  25. Catechol Dioxygenases Broderick,J.B.
  26. Structure v.7 Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions Sugimoto,K.;T.Senda;H.Aoshima;E.Masai;M.Fukuda;Y.Mitsui https://doi.org/10.1016/S0969-2126(99)80122-1