DOI QR코드

DOI QR Code

Lipolytic 효소를 생산하는 Acinetobacter sp. BD5 균주의 분리 및 특성

Isolation and Characterization of Acinetobacter sp. BD5 Producing Lipolytic Enzyme

  • 박인혜 (동아대학교 응용생명공학부) ;
  • 김선희 (동아대학교 응용생명공학부) ;
  • 이상철 (동아대학교 응용생명공학부) ;
  • 안순철 (부산대학교 의과대학 의학과) ;
  • 김철민 (부산대학교 의과대학 의학과) ;
  • 최용락 (동아대학교 응용생명공학부)
  • Park, In-Hye (Department of biotechnology, Faculty of Natural Resources and Life Science Dong-A University) ;
  • Kim, Sun-Hee (Department of biotechnology, Faculty of Natural Resources and Life Science Dong-A University) ;
  • Lee, Sang-Cheol (Department of biotechnology, Faculty of Natural Resources and Life Science Dong-A University) ;
  • Ahn, Soon-Cheol (School of medicine, College of Medicine, Pusan National University) ;
  • Kim, Cheol-Min (School of medicine, College of Medicine, Pusan National University) ;
  • Choi, Yong-Lark (Department of biotechnology, Faculty of Natural Resources and Life Science Dong-A University)
  • 발행 : 2006.07.31

초록

유용효소를 생산하는 균주를 가정하수로부터 분리하기 위하여 LBM, R2A, M9배지를 이용하여 다수의 균주를 분리하였다. 분리된 균주 중 1% tributyrin이 첨가된 배지에서 생육 활성대의 형성이 우수한 균주 1종을 최종적으로 선별하여 형태학적, 생리학적, 생화학적 특성을 관찰하였다. 16S rDNA 염기서열 분석결과 Acinetobacter Baumannii (99%)로 Acinetobacter 속에 속하는 균주임을 확인하고, Acinetobacter sp. BD5로 명명하였다. Acinetobacter sp. BD5는 $37^{\circ}C$$50^{\circ}C$에서 생육하는 것으로 보아 호열성 균주이며, 1% tributyrin과 oilve oil 첨가된 EL과 CE 고체배지와 Tween 20이 첨가된 LB 고체배지에서 생육활성대의 형성을 확인하여 이 균주가 lipolytic 효소를 생산하는 것으로 나타났다. 효소 활성의 최적 배양조건을 검토하고자 배양시간별 효소 활성을 측정한 결과, 배양 6시간 때인 대수 증식기에 가장 높은 효소활성을 나타내어 비교적 빠른 시간 내에 lipolytic 효소를 생성하는 것으로 나타났다. 또한 효소활성의 최적 온도는 $60^{\circ}C$$70-80^{\circ}C$에서 70%이상의 잔존활성을 보이는 것으로 나타나 Acinetobacter sp. BD5는 호열성 균주로 이 균주가 생산하는 lipolytic 효소도 내열성을 보이는 것으로 생각된다. 최적 pH는 9.0이며, pH 9.8-10.6범위에서 50% 이상의 활성이 유지되어 alkaline lipolytic 효소인 것으로 생각되어진다. Acinetobacter sp. BD5가 생산하는 lipolytic 효소는 비교적 넓은 pH 범위와 고온에서 활성이 유지되는 것으로 보아 폐유분해, 세제 합성과 유기물질 합성 등 생물공학분야와 산업적으로 잠재적 가치가 있을 것으로 생각된다.

A bacterium producing novel lipolytic enzyme was isolated from house sewage and identified as Acinetobacter sp. BD5 based on physiological characterization and 16S rDNA sequencing. The lipolytic activity of Acinetobacter sp. BD5 was tested using an EL agar medium and CE agar medium supplemented with 1% tributyrin and olive oil, respectively. The formation of a clear zone around the colony was detected by agar medium supplemented with 1% tributyrin and olive oil, respectively and Acinetobacter sp. BD5 formed powder-like zone around the colony on LB agar medium containing Tween 20. The quantitative lipolytic activity was determined by using p-NP butyrate as substrate. Acinetobacter sp. BD5 secreted the lipolytic enzyme during exponential growth phase, reaching a maximum amount after 6 hours of incubation. The lipolytic enzyme was found to be optimally active at $60^{\circ}C$ and retained more than 70% at $70-80^{\circ}C$. It displayed a high degree of activity in a pH of 7.0 to 10.6, with an optimal pH of 9.0.

키워드

참고문헌

  1. Bell, P. J. L., A. Sunna, M. D. Gibbs, N. C. Curach, H. Nevalainen and P. L. Bergquist. 2002. Prospecting for novel lipase genes using PCR. Microbiol. 148, 2283-2291 https://doi.org/10.1099/00221287-148-8-2283
  2. Han, S. J., J. H. Back, M. Y. Yoon, P. K. Shin, C. S. Cheong, M. H. Sung, S. P. Hong, I. Y. Chung and Y. S. Han. 2003. Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01. Biochimie. 85, 501-510 https://doi.org/10.1016/S0300-9084(03)00057-9
  3. Jaeger, K. E. and T. Eggert. 2002. Lipases for biotechnology. Curr. Opin. Biotechnol. 13, 390-397 https://doi.org/10.1016/S0958-1669(02)00341-5
  4. John, G. H., H. R. Krieg and P. H. A. Sneath. 1994. Bergey's maunal of systematic bacteriology:The proteobacteria partB the gammaproteobacteria. pp.425-437, 2th eds., Wiliams and Wilkins, Baltimore, USA
  5. Kim, H. E., I. S. Lee, J. H. Kim, K. W. Hahn, U. J. Park, H. S. Han and K. R. Park. 2003. Gene cloning, sequencing and expression of an esterase from Acinetobacter lwoffii I6C-1. Curr. Microbiol. 46, 91-295
  6. Kim, H. K., Y. J. Jung, W. C. Choi, H. S. Ryu, T. K. Oh and J. K. Lee. 2004. Sequence-based approach to finding functional lipases from microbial genome databases. FEMS Microbiol. lett. 235, 349-355 https://doi.org/10.1111/j.1574-6968.2004.tb09609.x
  7. Kok, R. G., C. B. Nudel, R. H. Gonzalez, I. M. N. Roodzant and K. J. Hellingwerf. 1996. Physiological factors affecting production of extracellular lipase(LipA) in Acinetobacter calcoaceticus BD413: Fatty acid repression of lipA expression and degradation lipA. J. bacterol. 178, 6025-6035 https://doi.org/10.1128/jb.178.20.6025-6035.1996
  8. Lee, D. G., C. M. Kim, S. J. Kim, S. H. Lee and J. H. Lee. 2003. Investigation of conserved regions in lipase genes. Kor. J. Life Science. 5, 723-731
  9. Lee, S. W., K. H. Woo, H. K. Lim, J. C. Kim, G. J. Choi and K. Y. Cho. 2004. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl. Microbiol. Biotechnol. 65, 720-726 https://doi.org/10.1007/s00253-004-1722-3
  10. Ma, J., Z. Zhang, B. Wang, X. Kong, Y. Wang, S. Cao and Y. Feng. 2006. Overexpression and characterization of a lipase from Bacillus subtilis. Protein Expr. Purif. 45, 22-29 https://doi.org/10.1016/j.pep.2005.06.004
  11. Mosbah, H., A. Sayari, H. Mejdoub, H. Dhouib, Y. Gargouri. 2005. Biochemical and molecular characterization of Staphylococcus xylosus lipase. Biochim. Biophys. Acta. 1723, 282-291 https://doi.org/10.1016/j.bbagen.2005.03.006
  12. Quyen, D. T., T. T. Nguyen, T. T. G. Le, H. K. Kim, T. K. Oh and J. K. Lee. 2004. A novel lipase/chaperone pair from Ralstonia sp. M1: analysis of the folding interaction and evidence for gene loss in R. solanacearum. Mol. Genet. Genomics. 272, 538-549 https://doi.org/10.1007/s00438-004-1084-7
  13. Rhee, J. K., D. G. Ahn., Y. G. Kim and J. W. Oh. 2005. New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl. Environ. Microbiol. 71, 817-825 https://doi.org/10.1128/AEM.71.2.817-825.2005
  14. Ruiz, C., F. I. J Poster and P. Diaz. 2003. Isolation and characterization of Bacillus sp. BP-6 LipA, a ubiquitous lipase among mesophilic Bacillus species. Lett. Appl. Microbiol. 37, 354-359 https://doi.org/10.1046/j.1472-765X.2003.01413.x
  15. Sekhon, A., N. Dahiya, R. P. Tiwari and G. S. Hoondal. 2005. Properties of a thermostable extracellular lipase from Bcillus megaterium AKG-1. J. Basic. Microbiol. 45, 147-154 https://doi.org/10.1002/jobm.200410498
  16. Sigurgrisladottir, S., M. Konraosdottir, A. Jonsson, J. K. Kristjansson and E. Matthiasson. 1993. Lipase activity of thermophilic bacteria from icelandic hot spring. Biotechnol. Lett. 15, 361-366 https://doi.org/10.1007/BF00128277
  17. Singh, Rajni, N. Gupta, V. K. Goswami and R. Gupta. 2004. A simple activity staining protocol for lipases and esterase. Appl. Microbiol. Biotechnol. 17, 1-4
  18. Snellman, E. A. and R. R. Colwell. 2004. Acinetobacter lipase: molecular biology, biochemical properties and biotechnological potential. J. Ind. Microbiol. Biotechnol. 31, 391-400 https://doi.org/10.1007/s10295-004-0167-0
  19. Sung, C. K., S. W. Gal, S. W. Lee and Y. J. Choi. 2001. Purification and characterization of extracellular lipase from Staphylococcus xylosus SC-22. Kor. J. Life Science. 11, 457-463
  20. Teo, J. W. P., L. H. Zhang and C. L. Poh. 2003. Cloning and characterization of a novel lipase from Vibrio harveyi strain AP6. Gene. 312, 181-188 https://doi.org/10.1016/S0378-1119(03)00615-2