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Journal of microbiology and biotechnology v.15 no.2, 2005년, pp.354 - 361   피인용횟수: 3

Molecular Cloning and Characterization of Alkaliphilic Phospholipase B (VFP58) from Vibrio fluvialis

AHN SUN HEE    (Department of Biotechnology and Bioengineering, Pukyong National University   ); JEONG SEUNG HA    (Department of Biotechnology and Bioengineering, Pukyong National University   ); KIM JIN MAN    (Division of Biotechnology and Chemical Engineering, Yosu National University   ); KIM YOUNG OK    (Biotechnology Research Center, National Fisheries Research and Development Institute   ); LEE SANG JUN    (Biotechnology Research Center, National Fisheries Research and Development Institute   ); KONG IN SOO    (Department of Biotechnology and Bioengineering, Pukyong National University  );
  • 초록

    Vibrio fluvialis, an enteropathogenic bacterium, produces a phospholipase which is thought to be an important factor in the pathogenesis of disease. In this study, the phospholipase gene (vfp) was identified from V fluvialis (KCTC 2473) and its sequence was determined. The entire open reading frame was composed of 1,689 nuc1eotides and 563 amino acids. The phospholipase gene (vfp) was overexpressed in Escherichia coli as a his-tag fused protein. This recombinant protein (rVFP58) was solubilized with 6 M urea and purified by Ni-NTA affinity chromatography. The action mode of rVFP58 was determined by TLC and GC-MS, and it showed phospholipase B activity, which had both phospholipase A and lysophospholipase activities. The rVFP58 showed a maximum activity at pH around 9- 10 and temperature of about 40OC, and it was stable under alkaline condition over pH 9. The cytotoxicity of rVFP58 was evaluated, using a fish cell line, CHSE-2l4, and was found to cause significant cell death after 14 h of exposure to 250 $\mu$ g of the protein.


  • 참고문헌 (42)

    1. Chen, S. C., L. C. Wright, R. T. Santangelo, M. Muller, V. R. Moran, P. W. Kuchcl, and T. C. Sorrell. 1997. Identification of extracellular phospholipase B, lyso phospholipase, and acyl transferase produced by Cryptococcus neoformans. Infect. lmmun. 65: 405-411 
    2. Flieger, A., S. Gongab, M. Faigle, H. A. Mayer, U. Kehrer, J. Mussotter, P. Bartmann, and B. Neumeister. 2000. Phospholipase A secreted by Leglonella pneumophila destroys alveolar surfactant phospholipids. FEMS Microhiol. Lett. 188: 129-133 
    3. Kume, N., M. I. Cybulsky, and M. A. Jr. Gimbrone. 1992. Lysophosphatidycholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J. Clin Invest. 90: 1138- 1144 
    4. Lee, J. H., S. H. Ahn, S. H. Kim, Y. H. Choi, K. J. Park, and I. S. Kong. 2002. Characterization of Vibrio tnimicus phospholipase A (PhlA) and cytotoxicity on fish cell. Biochem. Biophys. Res. Commun. 25: 269- 276 
    5. Lindahl, M., A. R. Hede, and C. Tagesson. 1986. Lysophosphatidylcholine increases airway and capillary permeability in the isolated perfused rat lung. Exp. Lung Res. 11: 1- 12 
    6. Mukherjee, P. K., K. R. Seshan, S. D. Leidich, J. Chandra, G. T. Cole, and M. A. Ghannoum. 2001. Reintroduction of the PLB1 gene into Candida albicans restores virulence in vivo. Microbiology 147: 2585- 2597 
    7. Prokazova, N. V., N. D. Zvezdina, and A. A. Korotaeva. 1998. Effect of lysophosphatidylcholme on transmembrane signal transduction. Biochemistry (Moscow) 63: 31- 37 
    8. Rahim, Z. and K. M. Aziz. 1996. Factors affecting production of haemolysin by strains of Vibrio fluvialis. J. Diarrhoeal Dis. Res. 14: 113- 116 
    9. Sanyal, S. C., R. K. Agarwal, E. Annapurna, and J. V. Lee. 1980. Enterotoxicity of group F vibrios. Jpn. J. Med. Sci. Biol. 33: 217 - 222 
    10. Sugiyama, Y., S. Nakashima, F. Mirbod, H. Kanoh, Y Kitajima, M. A. Ghannoum, and Y. Nozawa 1999. Molecular cloning of a second phospholipase B gene, caPLB2, from Candida albicans. Med. Mycol. 37: 61- 67 
    11. Yoon, S. C., J. H. Lee, S. H. Ahn, E. M. Lee. E. M. Park. and I. S. Kong. 2003. Purification and comparison of properties of the C-terminus truncated agarase of Pseudomonas sp. W7. J. Microbiol. Biotechnol. 13: 767-772 
    12. 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 
    13. Merkel, O., M. Fido, J. A. Mayr, H. Pruger, F. Raab, G. Zandonella, S. D. Kohlwein, and E Paltauf. 1999. Characterization and function in vivo of two novel phospholipases B/lysophospholipases from Saccharomyces cerevisiae. J. Biol. Chem. 274: 28121-28127 
    14. Lim, S. K., J. W. Choi, M. H. Chung, E. T. Lee, Y. H. Khang, S. D. Kim, and D. H. Nam. 2002. Production and characterization of extracellular phospholipase D from Streptomyces sp. YU100. J. Microbiol. Biotechnol. 12: 189-195 
    15. Cho, W. and F. J. Kczdy. 1991. Chromogenic substrates and assay of phospholipases $A_{2}$. Methods Enzymol. 195: 75- 79 
    16. Fifis, T., C. Costopoulos, and J. A. Vaughan. 1996. Evidence for phospholipase B activity in Fusobacterium necrophorum cultures and its association with hemolysinJIeucocidin activities. Vet. Microbiol. 49: 219- 233 
    17. Leidich, S. D., A. S. Ibrahim, Y. Fu, A. Koul, C. Jessup, J. Vitullo, W. Fonzi, F. Mibod, S. Nakashima, Y. Nozawa, and M. A. Ghannoum. 1998. Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans. J. Biol. Chem. 273: 26078- 26086 
    18. Dorrell, N., M. C. Martino, R. A. Stabler, S. J. Ward, Z. W. Zhang, A. A. McColm, M. J. Farthing, and B. W. Wren. 1999. Characterization of Helicobacter pylori P1dA. a phospholipase with a role in colonization of the gastric mucosa. Gastroenterology 117: 1098- 1104 
    19. Lee, J. H., Y. D. Cho, J. J. Choi, Y. J. Lee, H. S. Hoe, H. K. Kim, and S. T. Kwon. 2003. High-level expression in Escherichia coli of alkaline phosphatase from Thermus caldophilus GK24 and purification of the recombinant enzyme. J. Microbiol. Biotechnol. 13: 660- 665 
    20. Shim, E. J., S. H. Jeon, and K. H. Kong. 2003. Overexpression, purification, and biochemical characterization of the thermostable NAD-dependent alcohol dehydrogenase from Bacillus stearothermophilus. J. Microbiol. Biotechnol. 13: 738- 744 
    21. Singh, S., K. M. Cisera, J. D. Turnidge, and E. G. Russell. 1997. Selection of optimum laboratory tests for the identification of Moraxelia catarrhalis. Pathology 29: 206-208 
    22. Alam, M., S. Miyoshi, and S. Shinoda. 1995. Production of antigenically related exocellular elastolytic proteases mediating hemagglutination by vibrios. Microbiol. Immunol. 39: 67-70 
    23. Camilli, A., L. G. Tilney, and D. A. Portnoy. 1993. Dual roles of picA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8: 143-157 
    24. Baek, S. H., J. Y. Kwak, S. H. Lee, T. Lee, S. H. Ryu, D. J. Uhlinger, and J. D. Lambeth. 1997. Lipase activities of p37, the major envelope protein of vaccinia virus. J. Biol. Chem. 19: 32042- 32049 
    25. Nishijima, M., Y Akamatsu, and S. Nojima. 1974. Purification and properties of a membrane-bound phospholipase $A_{1}$ from Mycobacterium phlei. J. Biol. Chem. 249: 5658- 5667 
    26. Noverr, M. C., G. M. Cox, J. R. Perfect, and G. B. Huffnagle. 2003. Role of PLB1 in pulmonary inflammation and cryptococcal eicosanoid production. Infect. Immun. 71: 1538- 1547 
    27. Schmiel, D. H. and V. L. Miller. 1999. Bacterial phospholipases and pathogenesis. Microbes Infect. 1: 1103- 1112 
    28. Muckle, C. A. and C. L. Glyes. 1983. Relation of lipid content and exotoxin production to virulence of Corynebacterium pseudotuberculosis in mice. Am. J. Vet. Res. 44: 1149- 1153 
    29. Lee, J. V. and P. Shread. 1978. The taxonomy of group F organisms: Relationships to Vibrio and Aeromonas. J. Appl. Bacteriol. 45 ix 
    30. Timpe, J. M., M. M. Holm, S. L. Vanlerberg, V. Basrur, and E. R. Lafontaine. 2003. Identification of a Moraxella catarrhalis outer membrane protein exhibiting both adhesin and lipolytic activities. Infect. Immun. 71: 4341-4350 
    31. Lockwood, D. E., A. S. Kreger, and S. H. Richardson. 1982. Detection of toxins produced by Vibrio fluvialis. Infect. lmmun. 35: 702- 708 
    32. Niewoehner, D. E., K. Rice, A. A. Sinha, and D. Wangensteen. 1987. Injurious effects of lysophosphatidylcholinc on barrier properties of alveolar epithelium. J. Appl. Physiol. 63: 1979- 1986 
    33. Songer, J. G. 1997. Bacterial phospholipase and their role in virulence. Trends Microbiol. 5: 156- 161 
    34. Cox, G. M., H. C. McDade, S. C. Chen, S. C. Tucker, M. Gottfredsson, L. C. Wright, T. C. Sorrell, S. D. Leidich, A. Casadevall, M. A. Ghannoum, and J. R. Perfect. 2001. Extracellular phospholipase activity is a virulence factor for Cryptococcus neojormans. Mol. Microbiol. 39: 166- 175 
    35. Holm, B. A., L. Keicher, M. Y. Liu, J. Sokolowski, and G. Enhorning. 1991. Inhibition of pulmonary surfactant function by phospholipases. J. Appl. Physiol. 71: 317-321 
    36. Han, J. H., J. H. Lee, Y. H Choi, J. H. Park, T. J. Choi, and I. S. Kong. 2002. Purification, characterization and molecular cloning of vibrioftuvialis hemolysin. Biochim. Biophys. Acta 1599: 106- 114 
    37. Lockwood, D. E., S. H. Richardson, A. S. Kreger, M. Aliken, and B. Mc Creedy. 1983. In vitro and in vivo biologic activities of Vibrio fluvialis and its toxic products, pp. 87- 99. In S. Kawahara and N. E. Pierce (eds.), Advances in Research on Cholera and Related Diarrheas. Vol. 1. KTK Scientific Publishers. Tokyo, Japan 
    38. Mirbod, F., Y. Banno, M. A. Ghannoum, A. S. Ibrahim, S. Nakashima, Y. Kitajima, G. T. Cole, and Y. Nozawa. 1995. Purification and characterization of lysophosphoiipasetransacylase (h-LPTA) from a highly virulent strain of Candida albicans. Biochim. Biophys, Acta 1257: 181- 188 
    39. Aronson, J. F. and L. W. Johns. 1977. Injury of lung alveolar cells by lysolecithin. Exp. Mol. Pathol. 27: 35-42 
    40. Ghannourn, M. A. 2000. Potential role of phospholipase in virulence and fungal pathogenesis. Clin. Microbiol. Rev. 13: 122-143 
    41. Kothary, M. H., H. Lowman, B. A. McCardell, and B. D. Tall. 2003. Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis. Infect. lmmun. 71: 3213- 3220 
    42. Nishibuchi, M., R. J. Seidler, D. M. Rollins, and S. W. Joseph. 1983. Vibrio factors cause rapid fluid accumulation in suckling mice. Infect. Immun. 40: 1083-1091 
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    1. 2006. "" Journal of microbiology and biotechnology, 16(11): 1809~1813     
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