Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Purification and Characterization of Lipase from Acinetobacter sp. B2 Isolated from Oil­contaminated Soil

유류오염지역에서 분리한 Acinetobacter sp. B2로부터의 Lipase 정제 및 특성

  • 손성화 (한남대학교 이과대학 미생물학과) ;
  • 박경량 (한남대학교 이과대학 미생물학과)
  • Published : 2004.12.01
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Abstract

Three hundreds thirty two bacterial colonies which were able to degrade crude oil were isolated from soil sam­ples that were contaminated with oil in Daejeon area. Among them, one bacterial strain was selected for this study based on its higher oil degrading ability, and this selected bacterial strain was identified as Acinetobactor sp. B2 through physiological-biochemical tests and analysis of its 16S rRNA sequence. Acinetobactor sp. B2 was able to utilize various carbohydrates but did not utilize trehalose and mannitol as a sole carbon source. Acinetobactor sp. B2 showed a weak resistance to antibiotics such as kanamycin, streptomycin, tetracycline and spectinomycin, but showed a high resistance up to mg/ml unit to heavy metals such as Ba, Li, Mn, AI, Cr and Pb. The optimal growth temperature of Acinetobactor sp. B2 was $30^{\circ}C.$ The lipase produced by Acinetobactor sp. B2 was purified by ammonium sulfate precipitation, DEAE-Toyopearl 650M ion exchange chromatography and Sephadex gel filtration chromatography. Its molecular mass was about 60 kDa and condition for the optimal activity was observed at $40^{\circ}C$ and pH 10, respectively. The activation energy of lipase for the hydrolysis of p­nitrophenyl palmitate was 2.7 kcal/mol in the temperature range of 4 to $37^{\circ}C,$ and the enzyme was unstable at the temperature higher than $60^{\circ}C.$ The Michaelis constant $(K_m)\;and\;V_{max}$ for p-nitrophenyl palmitate were 21.8 uM and $270.3\;{\mu}M\;min^{-1}mg^{-1},$ respectively. This enzyme was strongly inhibited by 10 mM $Cd^{2+},\;Co^{2+},\;Fe^{2+},\;Hg^{2+},$ EDTA and 2-Mercaptoethalol.

대전 근교의 유류로 오염된 토양으로부터 유류를 분해하는332개의 세균 콜로니를 분리한 후 이 중 lipase 활성이 우수한 한 균주를 최종 선별하여 생리생화학적 조사와 16S rRNA 염기서열분석 등을 통하여 동정한 결과 Acinetobactor sp. B2로 확인되었다. 최종 선별된 Acinetobactor sp. B2는 trehalose, mannitol을 제외한 다양한 당을 이용하였고, kanamycin, streptomycin, tetracycline, spectinomycin의 항생제에 대해서 약한 내성을, 그리고 Ba, Li, Mn, Al, Cr, Pb 등의 중금속에 대해서는 mg/ml 단위까지 강한 내성을 나타냈고 생장 최적 온도는 $30^{\circ}C$로 확인되었다. Acinetobactor sp. B2에서 정제된 lipase의 분자량은60 kDa이었고, 이 효소의 최적 온도와 PH는 각각 $40^{\circ}C$와 pH 10이었다. 그리고p-nitrophenyl palmitate (pNPP)를 가수분해하는데 필요한 활성에너지는 $4-37{\circ}C$의 범위에서 2.7 kcal/mol 이었고, $60^{\circ}C$ 이상의 온도에서는 불안정한 효소임이 확인되었다. 또 pNPP에 대한 이 효소의 Michaelis constant (Km)와 최대속도상수 $(V_{max})$값은 각각 21.8 ${\mu}M$과 $270.3\;{\mu}M\;min^{-1}mg^{-1}$ 이었고, 이 효소는 $Cd^{2+},\;Co^{2+},\;Fe^{2+},\;Hg^{2+},$ EDTA, 2-mercaptoethanol에 의해 강하게 억제되었다.

Keywords

References

  1. 김갑정. 1999. Acinetobacter lwoffiid의 유류분해 및 생물학적 계면활성제의 특성. 한남대학교. 박사학위논문.
  2. 김혜은. 2001. Acinetobacter lwoffii I6C-1이 생산하는 Esterase 의 특성. 하남대학교. 석사학위논문.
  3. 윤남경, 박경량. 2004. 파라치온 분해 세균 Pseudomonas rhodesiae H5의 특성. 한국미생물학회지 14, 582-588
  4. Ahn, J.H., J.G. Pan, and J.S. Rhee. 1999. Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1. J. Bacteriol. 181, 1847-1852
  5. Atlas, R.M. 1975. Effects of temperature and crude oil composition on petroleum biodegradation. Appl. Microbiol. 30, 396-403
  6. Berto, P., L. Belingheri, and B. Dehorter. 1997. Production and purification of a novel extracellular lipase from Alternaria brassicicora. Biotechnology Lett. 19, 533-536
  7. Charusheela, A., and L. Arvind. 2002. Enzyme catalyzed hydrolysis of esters using reversibly soluble polymer conjugated lipases. Enzyme Microbial Technol. 30, 19-25
  8. Choo, D.W., T. Kurihara, T. Suzuki, K. Soda, and N. Esaki. 1998. A cold-adapted lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: gene cloning and enzyme purification and characterization. Appl. Environ. Microbiol. 64, 486-491
  9. Falk, M.P.F., E.A. Sanders, and W.D. Deckwer. 1990. Studies on the production of lipase from recombinant staphylococcus carnosus. Appl, Environ. Microbiol. 35, 10-13
  10. Floodgate, G.D. 1984. The fate of petroleum in marine ecosystem, In: R.M. Atlao (ed.), Macmilan Publishing co., New York. Colliet Macmillan publishers, London. 355-397
  11. Kim, G.J, I.S. Lee, and K.R. Park. 1999. Characteristics of Wasted Lubricant Degradation by Acinetobacter Lwoffii 16C-1. Kor. J. Life Science 9, 76-81
  12. Jobson, A., F.D. Cook, and D.W.S. Westlake. 1972. Microbial utilization of crude oil. Appl, Environ. Microbiol. 23, 1082-1089
  13. Kok, R.G., J.V.T. Jasper, M.N. Inge, B.W.B. Marc, R.E. Maarten, B.N. Clara, V. Ben, and J.H. Klaas. 1995. Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene. Molecular Microbiology 15, 803-818
  14. Kok, R.G., B.N. Clara, H.G. Rodrigo, M.N. Inge, and J.H. Klaas. 1996. Physiological factors affecting prduction of extracellular lipase(LipA) in Acinetobacter calcoaceticus BD413: Fatty acid repression of lipA expression and degradation of LipA. J. Bacteriol. 178, 6025-6035
  15. Krieg, N.R., and J.G. Holt. 1984. Bergey’s Manual of Systematic Bacteriology. Williams, Wilkins,and Baltimore
  16. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685
  17. Li, C.Y., C.Y. Cheng, and T.L. Chen. 2001. Production of Acinetobacter radioresistens lipase using Tween 80 as the carbon source. Enzyme Microbial Technol. 29, 258-263
  18. Lin, S.F., C.M. Chiou, C.M. Yeh, and Y.C. Tsai. 1996. Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111. Appl. Environ. Microbiol. 62, 1093- 1095
  19. Litthauer, D., A. Ginster, and E.V.E. Skein. 2002. Pseudomonas luteola lipase : A new member of the 320-residue Pseudomonas lipase family. Enzyme Microbial Technol. 30, 209-215
  20. Longshaw, C.M., A.M. Farrell, J.D. Wright, and K.T. Holland. 2000. Identification of a second lipase gene, gehD, in Staphylococcus epidermidis: comparison of sequence with those of other staphylococcal lipase, Microbiology 146, 1419-1427
  21. MacFaddin, J.F. 1984. Biochemical tests for identification of medical bacteria, 2nd ed. Williams & Wilkins CoMiget, R.J., C.H. Oppenheimer, H.I. Kator, and D.A. La. Rock. 1969. Microbial degradation of normal paraffin hydrocarbon in crude oil. In proceedings of the joint conference on prevention and control of oil spills, A.D.O.F.W.P.C.A. American Petroleum Institute., New York. 327-331
  22. Muralidhar, R.V., R.P. Chirumamilla, R. Marchant, V.N. Ramachandran, O.P. Ward and P. Nigam. 2002. Understanding lipase stereoselectivity. J. Microbial Biotech. Rev. 18, 81-97
  23. Nadini, M., D.A. Lang, K. Liebeton, K.E. Jaeger, and B.W. Dijkstra. 2000. Crystal structure of Pseudomonas aeruginosa lipase in the open conformation. J. Biol. Chem. 275, 31219-31225
  24. Oh, B.C., H.K. Kim, J.K. Lee, S.C. Kang, and T.K. Oh. 1999. Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning. FEMS. Microbial. Lett. 179, 385-392
  25. Organism central. 2001. Lippincott Williams and Wilkins Philadelphia. Baltimore. New York
  26. Pigne’de, G., W. Huijie, F. Franck, G. Claude, S. Michel, and N. Jean-Marc. 2000. Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica. J. Bacteriol. 182, 2802- 2810
  27. Reetz, M.T. 2002. Lipases as practical biocatalysts. Curr. Op. Chem. Biol. 6, 145-150
  28. Sharma, R., S.K. Soni, R.M. Vohra, L.K. Gupta, and J.K. Gupta. 2002. Purification and characterization of a thermostable alkaline lipase from a new thermophilic Bacillus sp. RSJ-1, Process Biochemistry 37, 1075-1084
  29. Sommer, P., C. Bormannm, and F. Gotz. 1997. Genetic and Bio chemical characterization of a New Extracellular Lipase from Streptomyces cinnamomeus, Appl. Environ. Microbio. 63, 3553- 3560
  30. Wagner, D.B., G.R. Furnier, M.A. Saghai-Maroof, S.M. Williams, B.P. Dancik, and R.W. Allard. 1987. Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc. Natl. Acad. Sci. USA. 84, 2097-2100 https://doi.org/10.1073/pnas.84.7.2097
  31. Wang, Y., K.C. Srivastava, G.J. Shen, and H.Y. Wang. 1995. Thermostable alkaline lipase from a newly isolated thermophilic Bacillus, strain A30-1(ATCC 53841). J. Fermentation Bioeng. 79, 433- 438
  32. Xuyang, L., T. Susanne, K.W. Ulrich, J. Karl-Erich, and J.B. Michael. 1995. Gene cloning, Sequence analysis, Purification, and Secretion by Escherichia coli of an Extracellular lipase from Serratia marcescens. Appl. Environ. Microbial. 61, 2674-2680