Identification and Cultural Characterization of Lipase Production Bacteria Isolated from Pond Effluent Sedimentary Layer

양식장 배출수 퇴적층에서 분리된 리파아제 생산 박테리아의 동정 및 배양학적 특성

  • Published : 2009.03.31

Abstract

From the course of screening of useful enzyme producing microorganism from marine sedimentary layer, we isolated 2 lipase producing strains and their lipase producing activities were tested. 16S rDNA sequence analysis showed that they were Gram-positive bacteria grouped on Janibacter sp. An excellent lipase producing strain, Janibacter sp. LI-68 and J. sp. LI-80 identified by 16S rDNA analysis and biochemical methods (BIOLOG), was further studied its lipase producing characteristics. The optimum initial pH, temperature and the optimum cultral time for the enzyme production on MA medium were 8, $30{\sim}40^{\circ}C$ and 96 h, respectively.

제주 연안 양식장 배출수 퇴적층으로부터 미생물 배양 배지를 사용하여 총 200여 균주를 분리하였으며, 분리된 균주를 이용하여 olive oil이 함유된 평판배지에서 투명환을 형성하는 균주를 분리하였고, 이를 LI-68, LI-80로 각각 명명하였다. 분리 균주 LI-68, LI-80의 BIOLOG를 이용한 생화학적 분석 특성 및 16S rDNA의 염기서열 분석 결과 Janibacter anophelis와 99%의 유전적 상동성을 보여 최종적으로 Janibacter sp. LI-68, Janibacter sp. LI-80으로 동정되었다. 분리 균주 Janibacter sp. LI-68과 Janibacter sp. LI-80의 증식을 위한 최적 배양온도를 확인하고 증식온도에 따른 지방분해효소 활성의 변화를 조사하였으며, 이를 위하여 배양 온도를 $20^{\circ}C$에서 $40^{\circ}C$까지 $10^{\circ}C$ 간격으로 배양하여 균체의 증식과 분비되는 지질분해 효소의 활성을 조사하였다. 그 결과 분리균주 Janibacter sp. LI-68는 $30^{\circ}C$ 배양실험구에서 가장 높은 균 생육도를 나타냈으며, 효소활성은 균 생육도와는 다른 $40^{\circ}C$에서 가장 높은 효소활성을 보였다. 반면 Janibacter sp. LI-80 균주는 $30^{\circ}C$에서 가장 높은 균 생육도를 보였으며, 효소활성은 균 생육도와 반비례하여 $40^{\circ}C$에서 가장 높은 효소활성을 보였다.

Keywords

References

  1. Bjorkling, F., S.E. Godtfredsen, and O. Kirt. 1991. The future impact of industrial lipases. Trends Biotechnol. 9, 360-363 https://doi.org/10.1016/0167-7799(91)90119-3
  2. Desnuelle, P. 1972. The lipases, pp. 575-616. In P.D. Boyer (ed.) Academic Press, New York and London
  3. Feller, G., E. Narinx, J.L. Arpigny, M. Aittaleb, E. Baise, S. Genicot, and C. Gerday. 1996. Enzymes from psychrophilic organisms. FEMS Microbiol. Rev. 18, 189-202 https://doi.org/10.1111/j.1574-6976.1996.tb00236.x
  4. Herbert, R.A. 1992. The perspective on the biotechnological potential of extremophiles. Trends Biotechnol. 10, 395-402 https://doi.org/10.1016/0167-7799(92)90282-Z
  5. Iwai, M., S. Okumura, and Y. Tsujsaka. 1975. The comparison of the properties of two lipases from Penicillin cyclopium westring. Agri. Biol. Chem. 39, 1063-1070 https://doi.org/10.1271/bbb1961.39.1063
  6. Jaeger, K.K., H.J. Choi, M.H. Kim, C.B. Sohn, and T.K. Oh. 2002. Expression and characterization of Ca$^2+$-dependent lipase from Bacillus pumilus B26. Biochim. Biophys. Acta. 1583, 205-212 https://doi.org/10.1016/S1388-1981(02)00214-7
  7. Jaeger, K.E., S.W. Dijkstra, and M.T. Reetz. 1999. Bacterial biocalysis: Molecular biology, three-dimensional structures, and biotechnological applications of lipase. Annu. Rev. Microbiol. 53, 315-351 https://doi.org/10.1146/annurev.micro.53.1.315
  8. Kim, J.W., S.Y. Shim, and S.S. Yoon. 1997. Isolation and purification of a lipase from Pseudomonas sp. YJ103 isolated from raw milk. Kor. J. Dairy Sci. 19, 17-24
  9. Kumar, S., K. Tamura, and M. Nei. 2004. Mega 3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinformatics 5, 150-163 https://doi.org/10.1093/bib/5.2.150
  10. Lee, J.M., R.S. Kim, B.O. Kim, Y.D. Park, and I.N. Jin. 1993. Isolation of a Pseudomonas aerusinosa strain producing an extracellular alkaline lipase catabolitely regulated by glucose, and purification of the Lipase. Kor. J. Appl. Microbiol. Biotechnol. 21, 161-168
  11. Lee, S.A., J.H. Lee, S.J. Kim, and H.K. Kim. 2005. Hydrolysis of triglyceride with cold-adapted lipase of Psychrobacter sp. S3 isolated from intertidal flat. Kor. J. Microbiol. Biotechnol. 33, 29-34
  12. Liu, W.A., T. Beppu, and K. Arima. 1973. Substrate specificity and mode of the lipase of thermophilic fungus Humicola lanuginosa S-38. Agri. Biol. Chem. 37, 1349-1355 https://doi.org/10.1271/bbb1961.37.1349
  13. Malcata, F.X. 1996. Engineering of/with lipases: Scope and strategies, pp. 1-16, Kluwer Academic Publishers, Netherlands
  14. Noble, L.D. and J.A. Gow. 1998. The effect of suspending solution supplemented with marine cations on the oxidation of Biolog GNMicroPlate$^TM$ substrates by Vibrionaceae bacteria. Can. J. Microbiol. 44, 251-258 https://doi.org/10.1139/cjm-44-3-251
  15. Otero, C., M.A. Berrendero, F. Cardenas, E. Alvarez, and S.W. Elson. 2005. General characterization of noncommercial microbial lipase in hydrolytic and synthetic reactions. Appl. Biochem. Biotechnol. 120, 209-223 https://doi.org/10.1385/ABAB:120:3:209
  16. Park, D.S., H.W. Oh, K.S. Bae, H.M. Kim, S.Y. Heo, N.J. Kim, K.Y. Seol, and H.Y. Park. 2007. Screening of bacteria producing lipase from insect gut: Isolation and characterization of a strain, Burkholderia sp. HY-10 producing lipase. Kor. J. Appl. Entomol. 46, 131-139 https://doi.org/10.5656/KSAE.2007.46.1.131
  17. Park, M.H., H.J. Ryu, and K.K. Oh. 2004. Isolation of lipase producing yeast and optimization of cultivation condition. Kor. J. Biotechnol. Bioeng. 19, 148-153
  18. Reetz, M.T. 2002. Lipases as practical biocatalysts. Curr. Opin. Chem. Biol. 6, 145-150 https://doi.org/10.1016/S1367-5931(02)00297-1
  19. Ren, T.J., F. Frank, and G.L. Christen. 1988. Characterization of lipase of Pseudomonas fluorescens 27; Based on fatty acid profiles. J. Dairy Sci. 71, 1432-1438 https://doi.org/10.3168/jds.S0022-0302(88)79705-2
  20. Ryu, H.S., H.K. Kim, W.C. Choi, M.H. Kim, S.Y. Park, N.S. Han, T.K. Oh, and J.K. Lee. 2006. New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases. Appl. Microbiol. Biotechnol. 70, 321-326 https://doi.org/10.1007/s00253-005-0058-y
  21. Sztajer, H. and E. Zboinsk. 1988. Microbial lipase in biotechnology. Acta Biotechnol. 2, 169-175 https://doi.org/10.1002/abio.370080211
  22. Taipa, M.A., M.R. Aires-Barros, and J.M.S. Cabral. 1992. Purification of lipase. J. Bacteriol. 26, 111-142 https://doi.org/10.1016/0168-1656(92)90001-P