Purification of Glucose Oxidase by Affinity Chromatography and Its Characterization

친화성 크로마토그래피를 이용한 글루코오스 옥시다아제의 정제와 효소특성

  • Ko Jung Hwan (Department of Biological Science and Engineering) ;
  • Byun Si Myung (Department of Biological Science and Engineering)
  • Published : 1979.06.30

Abstract

A purification technique of glucose oxidase was developed. Using the gluconyl-${\omega}$-aminohexyl Sepharose affinity chromatography, it was partially purified 14.6 folds with 79.7% yield. With the combination of the affinity chromatography and Sepharose 6B gel filtration, the enzyme was purified 27.2 folds from the broth with 74.1% yield. The final purified preparation showed 90.83 U of glucose oxidase activity per mg of protein and a single band by 7% polyacrylamide gel electrophoresis. The absorption spectrum and substrate specificity of the enzyme were studied and the fianal preparation showed the optimal pH between 5.6 and 6.0, the optimal temperature at $40^{\circ}C$, $8.5{\times}10^{-3}M$ of $K_m$ for D-glucose, and 3.43 kcal/mole of the activation energy.

토양중에서 분리한 Penicillium속이 생산하는 글루코오스 옥시타아제를 친화성 크로마토그래피에 의해 정제하고, 이 효소의 특성을 알아보았다. D-Gluconyl-${\omega}$-aminohexyl Sepharose컬럼을 사용하여 친화성 크로마토그래피를 행한 결과 14.6배 정제되었고 수율은 79%였으나 카탈라아제가 소량 함유되어 있어서 Sepharose 6B 겔 여과를 행하여 이를 제거하였다. 이 결과 27.2배 정제되고 수율이 74.1%인 정제효소를 얻었으며 7% polyacrylamide 겔 전기영동 결과 단일대를 보여주었고 비활성도는 단백질 mg당 90.83U였다. 정제된 효소의 흡광스펙트럼과 기질에 대한 특이성을 조사하였으며 최적 pH는 5.6∼6.0, 최적온도는 $40^{\circ}C$, D-글루코오스에 대한 $K_m$값은 $8.5{\times}10^{-3}M$, 활성화에너지는 3.43 kcal/mole이었다.

Keywords

References

  1. Biochem. Z. v.199 D. Muller
  2. Biochem. J. v.89 B. E. P. Swoboda;V. Massey;Q. H. Gibson;N. M. Atherson
  3. Biochemistry v.3 J. H. Pazur;K. Kleppe
  4. Biochem. J. v.39 C. E. Coulthard;R. Michaelis;W. F. Short;G. Sykes;G. E. H. Skrimshire;A. F. B. Standfast;J. H. Birkinshaw;H. Raistrick
  5. Biochem. J. v.42 D. Keilin;E. F. Hartree
  6. Biochem. Biophys. Acta v.40 K. Kusai;I. Sekuzu;B. Hagihara;K. Okunuki;S. Yamauchi;M. Nakai
  7. Ann. Rept. Sci. Works. Fac. Sci. Osaka Univ. v.8 K. Kusai
  8. Biochim. Biophys. Acta v.85 A. I. Schepartz;H. H. Subers
  9. Arch. Biochem. Biophys. v.91 E. C. Adams, Jr.;R. L. Mast;A. H. Free
  10. J. of Biochem. v.63 S. Nakamura;Y. Ogura
  11. J. Amer. Chem. Soc. v.91 F. R. Duke;M. Weibel;D. S Page;V. G. Bulgrin;J. Luthy
  12. Biochim. Biophys. Acta v.438 S. Hayashi;S. Nakamura
  13. Arch. Biochem. Biophys. v.103 J. H. Pazur;K. Kleppe; E. M. Ball
  14. Arch. Biochem. Biophys. v.111 J. H. Pazur;K. Kleppe;A. Cepure
  15. J. Biol. Chem. v.240 B. E. P. Swoboda;V. Massey
  16. Biochemistry Translated from Russian v.40 R. V. Valyulis;A. A. Glemzha;V. V. Trakimene
  17. Ms Thesis, KAIS J. H. Ko
  18. Enzyme, Enzyme Reagents and Related Biochemicals Worthington
  19. Methods of Enzymatic Analysis v.1 H. U. Bergmeyer;K. Gawehn;M. Grassl;H. U. Bergmeyer(Ed.)
  20. Boehringer mannheim mannual Biochemical Information I. Boehringer mannheim Co.
  21. J. Biol. Chem. v.193 O. H. Lowry;N. J. Rosebrough;A. L. Farr;W. H. Randall
  22. Anal. Biochem. v.81 R. Uy;F. Wold
  23. Anal. Biochem. v.60 S. C. March;I. Parikh;P. Cuatrecasas
  24. Anal. N. Y. Acad. Sci. v.121 R. J. Davis
  25. Affinity Chromatography C. R. Lowe;P. D. G. Dean