Journal of Microbiology and Biotechnology

  • 제15권6호
  • /
  • Pages.1189-1196
  • /
  • 2005
  • /
  • 1017-7825(pISSN)
  • /
  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Cloning and Characterization of Cyclohexanol Dehydrogenase Gene from Rhodococcus sp. TK6

  • CHOI JUN-HO (Division of Applied Biology and Chemistry, Kyungpook National University) ;
  • KIM TAE-KANG (Division of Applied Biology and Chemistry, Kyungpook National University) ;
  • KIM YOUNG-MOG (Institute of Agricultural Science & Technology, Kyungpook National University) ;
  • KIM WON-CHAN (Division of Applied Biology and Chemistry, Kyungpook National University) ;
  • JOO GIL-JAE (Institute of Agricultural Science & Technology, Kyungpook National University) ;
  • LEE KYEONG-YEOLL (Division of Applied Biology and Chemistry, Kyungpook National University) ;
  • RHEE IN-KOO (Division of Applied Biology and Chemistry, Kyungpook National University)
  • 발행 : 2005.12.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to $53\%$ with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.

키워드

참고문헌

  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402 https://doi.org/10.1093/nar/25.17.3389
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Brzostowicz, P. C., D. M. Walters, S. M. Thomas, V. Nagarajan, and P. E. Rouviere. 2003. mRNA differential display in a microbial enrichment culture: Simultaneous identification of three cyclohexanone monooxygenases from three species. Appl. Envir. Microbiol. 69: 334-342 https://doi.org/10.1128/AEM.69.1.334-342.2003
  4. Brzostowicz, P. C., K. L. Gibson, S. M. Thomas, M. S. Blasko, and P. E. Rouviere. 2000. Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display. J. Bacteriol. 182: 4241-4248 https://doi.org/10.1128/JB.182.15.4241-4248.2000
  5. Brzostowicz, P. C., M. S. Blasko, and P. E. Rouviere. 2002. Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU. Appl. Microbiol. Biotechnol. 58: 781-789 https://doi.org/10.1007/s00253-002-0968-x
  6. Cheng, Q., S. M. Thomas, K. Kostichka, J. R. Valentine, and V. Nagarajan. 2000. Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. strain SE19 by in vitro transposition. J. Bacteriol. 182: 4744-4751 https://doi.org/10.1128/JB.182.17.4744-4751.2000
  7. Choi, K. K., C. H. Park, S. Y. Kim, W. S. Lyoo, S. H. Lee, and J. W. Lee. 2004. Polyvinyl alcohol degradation by Microbacterium barkeri KCCM 10507 and Paenibacillus amylolyticus KCCM 10508 in dyeing wastewater. J. Microbiol. Biotechnol. 14: 1009-1013
  8. Donoghue, N. A. and P. W. Trudgill. 1975. The metabolism of cyclohexanol by Acinetobacter NCIB 9871. Eur. J. Biochem. 60: 1-7 https://doi.org/10.1111/j.1432-1033.1975.tb20968.x
  9. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmid. J. Mol. Biol. 166: 557-580 https://doi.org/10.1016/S0022-2836(83)80284-8
  10. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M. Kieser, D. J. Lydiate, C. P. Smith, and H. Schrempf. 1985. Genetic Manipulation of Streptomyces - A Laboratory Manual. The John Innes Foundation, Norwich, England
  11. Iwaki, H., Y. Hasegawa, M. Teraoka, T. Tokuyama, H. Bergeron, and P. C. K. Lau. 1999. Identification of a transcriptional activator (ChnR) and a 6-oxohexanoate dehydrogenase (ChnE) in the cyclohexanol catabolic pathway in Acinetobacter sp. strain NCIMB 9871 and localization of the genes that encode them. Appl. Environ. Microbiol. 65: 5158-5162
  12. Jin, H. H., N. S. Han, D. H. Kweon, Y. C. Park, and J. H. Seo. 2001. Effects of environmental factors of in vivo folding of Bacillus macerans cyclodextrin glycosyltransferase in recombinant Escherichia coli. J. Microbiol. Biotechnol. 11: 92-96
  13. Jornvall, H., B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J. Jeffery, and D. Ghosh. 1995. Shortchain dehydrogenases/reductases (SDR). Biochemistry 34: 6003-6013 https://doi.org/10.1021/bi00018a001
  14. Kim, Y. M., K. Park, J. H. Choi, J. E. Kim, and I. K. Rhee. 2004. Biotransformation of the fungicide chlorothalonil by bacterial glutathione S-transferase. J. Microbiol. Biotechnol. 14: 938-943
  15. Kim, T. K. and I. K. Rhee. 1999. Cyclohexanol dehydrogenase isozymes produced by Rhodococcus sp. TK6. Kor. J. Appl. Microbiol. Biotechnol. 27: 124-128
  16. Kim, T. K. and I. K. Rhee. 1999. Isolation and characterization of cyclohexanol utilizing bacteria. Kor. J. Appl. Microbiol. Biotechnol. 27: 107-112
  17. Kim, T. K., J. H. Choi, and I. K. Rhee. 2002. Purification and characterization of a cyclohexanol dehydrogenase from Rhodococcus sp. TK6. J. Microbiol. Biotechnol. 12: 39-45
  18. Kwon, H. H., E. Y. Lee, K. S. Cho, and H. W. Ryu. 2003. Benzene biodegradation using the polyurethane biofilter immobilized with Stenotrophomonas maltophilla T3-c. J. Microbiol. Biotechnol. 13: 70-76
  19. Neidle, E. L., C. Hartnett, N. L. Ornston, A. Bairoch, M. Rekik, and S. Harayama. 1992. Cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily. Eur. J. Biochem. 204: 113-120 https://doi.org/10.1111/j.1432-1033.1992.tb16612.x
  20. Park, H. D., G. J. Joo, and I. K. Rhee. 1997. Overproduction of Escherichia coli D-xylose isomerase using ${\lambda}P_L$ promoter. J. Microbiol. Biotechnol. 7: 8-12
  21. Park, M. K., K. H. Liu, Y. H. Lim, Y. H. Lee, H. G. Hur, and J. H. Kim. 2003. Biotransformation of a fungicide ethaboxam by soil fungus Cunninghamella elegans. J. Microbiol. Biotechnol. 13: 43-47
  22. Persson, B., M. Krook, and H. Jornvall. 1991. Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur. J. Biochem. 200: 537-543 https://doi.org/10.1111/j.1432-1033.1991.tb16215.x
  23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., U.S.A
  24. Sanger, F. S., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467
  25. Schlieker, C., B. Bukau, and A. Mogk. 2002. Prevention and reversion of protein aggregation by molecular chaperones in E. coli cytosol: Implications for their applicability in biotechnology. J. Biotechnol. 96: 13-21 https://doi.org/10.1016/S0168-1656(02)00033-0
  26. Shin, C. S., M. S. Hong, H. C. Shin, and J. W. Lee. 2001. High-level production of recombinant human IFN-${\alpha}2a$ with co-expression of $tRNA^{Arg(AGG/AGA)}$ in high-cell-density cultures of Escherichia coli. Biotechnol. Bioprocess. Eng. 6: 301-305 https://doi.org/10.1007/BF02931994
  27. Stirling, L. A. and J. J. Perry. 1980. Purification and properties of a nicotinamide adenine dinucleotide-linked cyclohexanol dehydrogenase from a Nocardia species. Curr. Microbiol. 4: 37-40 https://doi.org/10.1007/BF02602889
  28. Tanaka, H., H. Obata, T. Tokuyama, T. Ueno, F. Yoshizako, and A. Nishmura. 1977. Metabolism of cyclohexanol by Pseudomonas species. Hakkokogaku Kaishi 55: 62-67
  29. Thomas, J. G. and F. Baneyx. 1997. Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli. Protein Expr. Purif. 11: 289-296 https://doi.org/10.1006/prep.1997.0796
  30. Trower, M. K., M. Buckland, R. Higgins, and M. Griffin. 1985. Isolation and characterization of cyclohexane-metabolizing Xanthobacter sp. Appl. Environ. Microbiol. 49: 1282-1289
  31. Van Beilen, J. B., F. Mourlane, M. A. Seeger, J. L. Z. Kovac, T. H. Smits, U. Fritsche, and B. Witholt. 2003. Cloning of Baeyer-Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ. Microbiol. 5: 174-182 https://doi.org/10.1046/j.1462-2920.2003.00401.x
  32. Vieira, J. and J. Messing. 1987. Production of singlestranded plasmid DNA. Methods Enzymol. 153: 3
  33. Whyte, L. G., T. H. M. Smits, D. Labbe, B. Witholt, C. W. Greer, and J. B. van Beilen. 2002. Cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus spp. strains Q15 and 16531. Appl. Environ. Microbiol. 68: 5933-5942 https://doi.org/10.1128/AEM.68.12.5933-5942.2002