Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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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)
  • Published : 2005.04.01
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Abstract

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.

Keywords

References

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