Purification and Characterization of Catechol 2,3-Dioxygenase from Recombinant Strain E. coli CNU312.

재조합균주 E. coli CNU312가 생산하는 Catechol 2,3-Dioxygenase의 정제 및 특성

  • Published : 2000.03.01

Abstract

Catechol 2,3-dioxygenase was purified from recombinant strain E. coli CNU312 carrying the tomB gene which was cloned from toluene-degrading Burkholderia cepacia G4. The purification of this enzyme was performed by acetone precipitation, Sephadex G-75 chromatography, electrophoresis and electro-elution. The molecular weight of native enzyme was about 140.4 kDa and its subunit was estimated to be 35 kDa by SDS-PAGE. It means that this enzyme consists of four identical subunits. This enzyme was specifically active to catechol, and$K_(m)$ value and $V_(max)$value of this enzyme were 372.6 $\mu$M and 39.27 U/mg. This enzyme was weakly active to 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol, but rarely active to 2,3-DHBP. The optimal pH and temperature of the enzyme were pH 8.0 and $40^{\circ}C$. The enzyme was inhibited by $Co^(2+)$, $Mn^(2+)$, $Zn^(2+)$, $Fe^(2+)$, $Fe^(3+)$, and $Cu^(2+)$ ions, and was inactivated by adding the reagents such as N-bromosuccinimide, and $\rho$-diazobenzene sulfonic acid. The activity of catechol 2,3-dioxygenase was not stabilized by 10% concentration of organic solvents such as acetone, ethanol, isopropyl alcohol, ethyl acetate, and acetic acid, and by reducing agents such as 2-mercaptoethanol, dithiothreitol, and ascorbic acid. The enzyme was inactivated by the oxidizing agent $H_(2)$$O_(2)$, and by chelators such as EDTA, and ο-phenanthroline.

Toluene, phenyl 등의 분해균주인 Burkholderia cepacia G4로부터 tomB 유전자를 클로닝하여 얻은 재조합 균주 E. coli CNU312로부터 catechol 2,3-dioxygenase를 정제하여 효소학적 특성을 조사하였다. Catechol 2,3-dioxygenase는 native 분자량이 약 140.4 kDa이었으며 4개의 동일한 35 kDa subunit로 구성된 homotetramer로 생각된다. Catechol의 $K_(m)$값과 $V_(max)$값은 372.6 $\mu$M과 39.27 U/mg이었으며, 1.56 mM 이상의 기질 농도에서는 활성이 감소되었다. 효소 활성의 최적 pH는 8.0이었으며, pH 7.0-8.0 범위에서 안정하였다. 최적 활성온도는 $40^{\circ}C$였으며, $60^{\circ}C$이상에서 완전히 활성을 상실하였다. 또한 $Fe^(2+)$, $Fe^(3+)$ 를 비롯한 대부분의 금속 이온에 의해 활성이 감소되었으며, $Mg^(2+)$, $K^(+)$에는 영향을 받지 않았다. 효소 활성부위를 알아보기 위해 화학변형제를 처리한 결과, tryptophan과 histidine이 효소 활성부위에 존재하는 것으로 추정된다. 그리고 10%의 유기용매에 안정성을 보이지 않았으며, $H_(2)$$O_(2)$, EDTA, ο-phenanthroline에도 활성이 감소되었다. 또한 2-mercaptoethanol, dithiothreitol, 그리고 ascorbic acid와 같은 환원제에 대해서도 안정성을 보이지 않았다. 이 효소는 catechol에 대해 높은 기질 특이성을 보였으며, 3-methylcatechol, 4-methylcatechol, 그리고 4-chlorocatechol에 대해 약간의 활성을 보였다. 그러나 2,3-dihydroxybiphenyl에 대해서는 거의 활성을 보이지 않았다.

Keywords

References

  1. J. Biol.Chem v.269 Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6 Asturias, J. A.;L. D. Eltis;M. Prucha;K. N. Timmis
  2. J. Biol. Chem v.268 Purification and crystalization of 2,3-dihydroxybiphenyl 1,2-dioxygenase Eltis, L. D.;B. Hofmann;H. J. Hecht;H. Lunsdorf;K. N. Timmis
  3. J. Bacteriol v.169 Purification and properties of 2,3-dihydroxybiphenyl dioxygenase from polychlorinated bi-phenyl-degrading Pseudomonas pseudoalcaligenes and Pseudomonas aeruginasa carrying the cloned bph C gene Furukawa, K.;N. Arimura
  4. J. Biol. Chem v.264 Bacterial aromatic ring-cleavage enzyme are classified into two different gene famlies Harayama, S.;M. Rekik
  5. FEMS Microbiol. Lett. v.118 Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin Hirose, J.:N. Kimura;A. Suama;A. Kobayash;S. Hayashida;K. Furukawa
  6. J. Bacteriol v.180 Degradation of chloroaromatics: purification and characterization of a novel type of cholrocatechol 2,3-dioxygenase of Pseudomonas putida GJ31 Kaschabek, S.;T. Kasberg;D. Muller;A. E. Mars;D. B. Janssen;W. Reineke
  7. Kor. J. Microbiol v.25 Isolation and characterization of bacteria degrading chlorinated aromatic hydrocarbons Kim, J. W.;C. K. Kim;Y. C. Kim;J. H. Yeoum;J. G. Lee
  8. Kor. J. Microbiol v.35 Characteristics of catechol 2,3-dioxygenase producted by 4-chlorobenzoate-degrading Pseudomonas sp.S-47 Kim, K. P.D. I. Seo;K. H. Min;J. O. Ka;Y. K. Park;C. K. Kim
  9. Biochem. Biophys. Res. Commun v.224 Structure of catechol 2,3-dioxygenase gene encoded in chromosomal DNA of Pseudomonas putida KF715 Lee, J. R.;Y. C. Kim;J. Y. Lim;Y. S. Kim
  10. Appl. Environ Microbiol v.47 Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorianted biphenyls Masse, R.;F. Messier;L. Peloquin;C. Ayotte;M. Sylvestre
  11. Biochem. Biophys. Res. Commun v.238 Characterization of the gene encoding catechol 2,3-dioxygenase from Achromobacter xylosoxidans KF701 Moon, J. H.;E. J. Kang;K. R. Min;C. K. Kim;K. H. Min;K. S. Lee;Y. S. Kim
  12. Biochem. Biophys. Res. Commun v.234 no.3 Structure of catechol 2,3-dioxygenasegene encoded in TOM plasmid of Pseudomonas cepacia G4 Oh, J. M.;K. R. Min;C. K. Kim;Y. C. Kim;J. Y. Lim;K. S. Lee;K. H. Min;Y. S. Kim
  13. Kor, J. Appl. Microbiol. Biotechnol v.24 Purification and characterization of 2,3-dihydroxybiphenyl dioxygenase from recombinant E. coli CK 1092 Park, H. N.;Y. S. Kim;Y. C. Kim;C. K. Kim;J. Y. Lim
  14. J. Biocteriol. v.179 Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. stain DPO360 Schmid, A.;B. Rothe, J.;Altenbuchner;W. Ludwig;K. H. Engesser
  15. 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
  16. Appl. Environ. Microbiol. v.39 Isolation method for bacterial isolates capable of growth on p-chlorobiphenyl Sylvestre, M.
  17. J. Bacteriol. v.175 Purification and characterization of protocatechol 2,3-dioxygenase from Bacillus maceransia new extradiol catecholic dioxygenase Wolgel, S. A.;J. E. Dega;P. E. Perkins-Olson;C. H. Juarez-Garicia;R. L. Crawford;E. $M\"{u}nck$;J. D. Lipscomb