Studies on Pyrocatechase from a Soil Bacterium (Ⅰ). Purification and Characterization of Pyrocatechase

토양 박테리아로부터의 Pyrocatechase 에 관한 연구 (제1보). 효소정제와 특성연구

  • Yeon-Bo Chung (Department of Biological Science and Engineering, Korea Advanced Institute of Science) ;
  • Hyun-Jae Lee (Department of Biological Science and Engineering, Korea Advanced Institute of Science)
  • 정연보 (한국과학원 생물공학과) ;
  • 이현재 (한국과학원 생물공학과)
  • Published : 19800230

Abstract

Pyrocatechase as a phenolytic dioxygenase was extracted from the benzoate-induced cells of a soil bacterium, a member of Pseudomonadaceae, and purified partially by DEAE-cellulose ion-exchange chromatography and Sephadex G-75 gel filtration. Final preparation of the enzyme yielding 200 fold purification over the crude extracts showed a specific activity of about 40 ${\mu}moles$ per minute per mg protein based on catechol as the substrate. The enzyme showed a very limited substrate specificity towards catechol for its catalytic activity. Based on the inhibition study with the substrate analogues, it was assumed that ortho dihydroxy groups on the aromatic ring may participate in the enzyme-substrate binding. The $K_m$ value for catechol was obtained as $1.9{\times}10^{-6}M$, and the optimum activity of the enzyme was obtained at the pH range of 7∼10 and $35^{\circ}C$. With SH-group blocking agents the enzyme was inhibited seriously. The activity of enzyme was also inhibited by the addition of some heavy metals, $Ag^+$ and $Cu^{2+}$, but was not affected by EDTA. General property of the enzyme was characterized and the possible nature of the enzyme active center was also discussed.

Keywords

References

  1. Molecular Mechanisms of Oxygen Activation M. Nozaki;O. Hayaishi(ed.)
  2. J. Biochem. (Tokyo) v.37 O. Hayaishi;K. Hashimoto
  3. J. Amer. Chem. Soc. v.77 O. Hayaishi;M. Katagiri;S. Rothberg
  4. J. Amer. Chem. Soc. v.77 H. S. Mason;W. L. Fowlks;E. Peterson
  5. J. Bact. v.100 C. G. Feist;G. D. Hegeman
  6. J. Bact. v.102 R. Y. Stanier;D. Wachter;C. Gasser
  7. J. Biochem. (Tokyo) v.66 H. Kita;Y. Miyake;M. Kamimoto;S. Senoh;T. Yamano
  8. J. Amer. Chem. Soc. v.79 O. Hayaishi
  9. Biochem. Biophys. Res. Comm. v.47 K. Nogami
  10. The Enzyme v.ⅩⅡ O. Hayaishi;M. Nozaki;M. T. Abbot;P. D. Boyer(ed.)Ⅹ
  11. Methods in Enzymology v.Ⅰ O. Haiyaishi;S. P. Colowick(ed.);N. O. Kaplan(ed.)
  12. Bergey's Manual of Determinative Bacteriology R. E. Buchanan;N. E. Gibbons(eds.)
  13. Laboratory Methods in Food and Dairy Microbiology W. E. Harrigan;M. E. McCance
  14. Principles of Microbe and Cell Cultivation S. J. Prit
  15. J. Biol. Chem. v.242 Y. Kojima;H. Fujisawa;A. Nakazawa;T. Nakazawa;F. Kanetsuna;H. Taniuchi;M. Nozaki;O. Hayaishi
  16. J. Biol. Chem. v.193 O. H. Lowry;N. J. Rosebrough;A. L. Farr;R. J. Randall
  17. Methods in Enzymology v.ⅩⅩⅡ P. McPhie;W. B. Jakoby(ed.)
  18. Fed. Proc. v.23 H. Taniuchi;Y. Kojima;A. Nakazawa;O. Hayaishi
  19. Process Biochem. v.13 no.6 H. Y. Neujahr
  20. J. Biol. Chem. v.229 O. Hayaishi;M. Katagiri;S. Rothberg
  21. Essays in Biochemistry v.Ⅱ S. Dagley;P. N. Campbell(ed.);W. N. Aldridge(ed.)
  22. Biological and Chemical Aspects of Oxygenases M. Nozaki;Y. Kojima;T. Kakazawa;H. Fujisawa;K. Ono;S. Kotani;O. Hayaishi;T. Yamano;K. Bloch(ed.);O. Hayaishi(ed.)
  23. Biochem. Biophys. Res. Comm. v.42 K. Nogami;Y. Miyake
  24. Science v.164 O. Hayaishi;M. Nozaki
  25. J. Bact. v.127 R. N. Pater;C. T. Hou;A. Feux;M. O. Lillard
  26. Appl. Envirn. Microbiology v.33 no.3 C. T. Hou;R. Patel;M. O. Lillard