The Expression and Functional Analysis of Recombinant Alcohol Dehydrogenase

재조합 alcohol dehydrogenase의 발현 및 기능분석

  • Received : 1999.04.29
  • Published : 1999.12.25

Abstract

The alcohol dehydrogenase (ADH) gene from Bacillus stearothermopilus was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pGEX-KG, and expressed it as a fusion protein with glutathione S-transferase (GST) in E. coli. The recombinant ADH was produced by induction with 1 mM isopropyl-${\beta}$-D-thiogalactopyranoside at $37^{\circ}C$ and purified by glutathione affinity chromatography. The recombinant ADH exhibited high substrate specificity for ethanol. The activity of the recombinant ADH proceeded optimally at pH 9.0 and $70^{\circ}C$. The recombinant ADH was highly stable against high temperature. This thermostable alcohol dehydrogenase can be used for the enzymatic determination of alcohol and for the industrial production of alcohol.

Keywords

alcohol dehydrogenase;Bacillus stearothermopilus;expression in E. coli

References

  1. Biochem. Pharmacol. v.27 R. Pietruszko;K. Voigtlander;D. Lester
  2. Plant Science v.107 U. Oluoha
  3. Biochem. Biophys. Res. Commum. v.211 K. M. Madyastha;T. L. Gururaja
  4. Biochem. J. v.303 I. Bruchhaus;E. Tannich
  5. Eur. J. Biochem. v.120 J. Jeffery;L. Cummins;M. Carlqust;H. Jornvall
  6. Eur. J. Biochem. v.154 A. D. Neale;R. K.copes;J. M. Kelley;R. E. H. Wettenhall
  7. Biosens. Bioelectron. v.6 J. Zhao;R. P. Buck
  8. Anal. Lett. v.27 M. Gotoh;I. Karube
  9. Biosens. Bioelectron. v.6 S. Miyamoto;T. Murakami;A. Saito;J. Kimura
  10. Anal. Biochem. v.192 K. L. Guan;J. E. Dixon
  11. Eur. J. Biochem. v.222 R. Cannio;M. Rossi;S. Bartolucci
  12. Plant Cell Reports v.15 S. M. Li;Z. X. Wang;L. Heide
  13. J. Bacteriol. v.175 C. M. Hansgens;J. Vonck;J. Van Beeumen;E. F. Van Bruggen;T. A. Hansen
  14. J. Bacteriol v.174 H. Sakoda;T. Imanaka
  15. Shin Seigakaku jikken (Tokyo) v.1 R. Takenoshita;T. Togi
  16. J. Bacteriol. v.175 A. A. Ismaiel;C. X. Zhu;G. D. Colby;J. S. Chen
  17. Nature (London) v.227 U. K. Laemmli
  18. Eur. J. Biochem. v.141 A. Steinbuchel;H. G. Schlegel
  19. Anal. Biochem. v.72 M. M. Bradford
  20. Plant Cell Reports v.15 S. M. Li;Z. X. Wang;L. Heide
  21. Biochem. Pharmacol. v.28 L. Lumeng;W. F. Bosron;T. K. Li
  22. J. Biol. Chem. v.268 D. W. Green;H. W. Sun;B. V. Plapp
  23. Eur. J. Biochem. v.222 K. N. Truscott;P. B. Hoj;R. K. Scopes

Acknowledgement

Supported by : 과기부