Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Purification and Characterization of a Cyclohexanol Dehydrogenase from Rhodococcus sp. TK6

  • Kim, Tae-Kang (Department of Agricultural Chemistry, Kyungpook National University) ;
  • Choi, Jun-Ho (Department of Agricultural Chemistry, Kyungpook National University) ;
  • Rhee, In-Koo (Department of Agricultural Chemistry, Kyungpook National University)
  • Published : 2002.02.01
Download PDF
⟨ Previous Next ⟩

Abstract

Activity staining on the native polyacrylamide gel electrophoresis (PAGE) of a cell-free extract of Rhodococcus sp. TK6, grown in media containing alcohols as the carbon source, revealed at least seven isozyme bands, which were identified as alcohol dehydrogenases that oxidize cyclohexanol to cyclohexanone. Among the alcohol dehydrogenases, cyclohexanol dehydrogenase II (CDH II), which is the major enzyme involved in the oxidation of cyclohexanol, was purified to homogeneity. The molecular mass of the CDH II was determined to be 60 kDa by gel filtration, while the molecular mass of each subunit was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The CDH II was unstable in acidic and basic pHs, and rapidly inactivated at temperatures above $40^{\circ}C$ . The CDH II activity was enhanced by the addition of divalent metal ions, like $Ba^2+\;and\;Mg^{2+}$. The purified enzyme catalyzed the oxidation of a broad range of alcohols, including cyclohexanol, trans-cyclohexane-1,2-diol, trans-cyclopentane-l,2-diol, cyclopentanol, and hexane-1,2-diol. The $K_m$ values of the CDH II for cyclohexanol, trans-cyclohexane-l,2-diol, cyclopentanol, trans-cyclopentane-l,2-diol, and hexane-l,2-diol were 1.7, 2.8, 14.2, 13.7, and 13.5 mM, respectively. The CDH II would appear to be a major alcohol dehydrogenase for the oxidation of cyclohexanol. The N-terminal sequence of the CDH II was determined to be TVAHVTGAARGIGRA. Furthermore, based on a comparison of the determined sequence with other short chain alcohol dehydrogenases, the purified CDH II was suggested to be a new enzyme.

Keywords

References

  1. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Bradford, M. M. https://doi.org/10.1016/0003-2697(76)90527-3
  2. J. Bacteriol. v.182 Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. strain SE19 by in vitro transposition Cheng, Q.;S. M. Thomas;K. Kostichka;J. R. Valentine;V. Nagarajan https://doi.org/10.1128/JB.182.17.4744-4751.2000
  3. Ann. N. Y. Acad. Sci. v.121 Disc electrophoresis Ⅱ; Method and application to human serum proteins Davis, B. J. https://doi.org/10.1111/j.1749-6632.1964.tb14213.x
  4. Arch. Microbiol. v.153 Enzyme reactions involved in anaerobic cyclohexanol metabolism by a denitrifying Pseudomonas species Dangel, W.;A. Tschech;G. Fuchs
  5. Eur. J. Biochem. v.74 The metabolism of trans-cyclohexane-1,2-diol by an Acinetobacter species Davey, J. F.;P. W. Trudgill https://doi.org/10.1111/j.1432-1033.1977.tb11373.x
  6. Eur. J. Biochem. v.60 The metabolism of cyclohexanol by Acinetobacter NCIB 9871 Donoghue, N. A.;P. W. Trudgill https://doi.org/10.1111/j.1432-1033.1975.tb20968.x
  7. Eur. J. Biochem. v.1 A protein sequenator Edman, P.;G. Begg
  8. Kor. J. Appl. Microbiol. Biotechnol. v.27 Isolation and characterization of cyclohexanol-utilizing bacteria Kim, T. K.;I. K. Rhee
  9. Kor. J. Appl. Microbiol. Biotechnol. v.27 Cyclohexanol dehydrogenase isozymes produced by Rhodococcus sp. TK6 Kim, T. K.;I. K. Rhee
  10. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T₄+ Laemmli, U. K. https://doi.org/10.1038/227680a0
  11. Eur. J. Biochem. v.204 Cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xyIL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily Neidle, E. L.;C. Hartnett;N. L. Ornston;A. Bairoch;M. Rekik;S. Harayama https://doi.org/10.1111/j.1432-1033.1992.tb16612.x
  12. Biochem. J. v.121 The metabolism of cyclohexanol by Nocardia globerula CL1 Norris, D. B.;P. W. Trudgill https://doi.org/10.1042/bj1210363
  13. Eur. J. Biochem. v.174 Purification and properties of 4-hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum Obata, H.;M. Uebayasi;T. Kaneda https://doi.org/10.1111/j.1432-1033.1988.tb14119.x
  14. Recombinant DNA Techniques Rodriguez, R. L.;R. C. Trait
  15. Nature v.250 Chemical and biological evolution of a nuelectide-binding protein Rossmann, G. M.;D. Moras;K. W. Olsen https://doi.org/10.1038/250194a0
  16. Curr. Microbiol. v.4 Purification and properties of a nicotinamide adenine dinucleotide-linked cyclohexanol dehydrogenase from a Nocardia species Stirling, L. A.;J. J. Perry https://doi.org/10.1007/BF02602889
  17. Appl. Environ. Microbiol. v.66 Thermostable NADP+-dependent medium-chain alcohol dehydrogenase from Acinetobacter sp. strain M-1: Purification and characterization and gene expression in Escherichia coli Tani, A.;Y. Sakai;A. Ishige;K. Nobuo https://doi.org/10.1128/AEM.66.12.5231-5235.2000
  18. Appl. Environ. Microbiol. v.49 Isolation and characterization of a cyclohexane-metabolizing Xanthobacter sp. Trower, M. K.;R. M. Buckland;R. Higgins;M. Griffin
  19. Microbial Degradation of Organic Compounds Microbial degradation of the alicyclic ring. Structural relationships and metabolic pathways Trudgill, P. W.;D. T. Gibson(ed.)
  20. J. Biol. Chem. v.274 Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short-chain dehydrogenase/reductase superfamily Van der Werf, M. J.;C. van der Ven;F. Barbirato;M. H. M. Eppink;J. A. M. de Bont;W. J. H. van Berkel https://doi.org/10.1074/jbc.274.37.26296