Abstract
A phospholipase (PLase) gene of Vibrio vulnificus was cloned in Escherichia coli and the properties of the gene product were investigated. The PLase structural gene was composed of 1,251 bp, encoding 417 amino acids for a protein with a predicted molecular mass of 47,187 Da including a putative signal sequence. The predicted protein sequence was 87 and 82% identical to those of hemolysins from Vibrio spp. and that of lecithinase from V. cholerae, respectively. A lipid binding motif, GDSL, conserved among various PLases and lipases was also observed. Over-expression of PLase caused inclusion body formation in E. coli, but not that of the PLase subclone without the signal sequence (45 kDa). Purified PLase exhibited hemolytic activity on red blood cells and hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and soya-lecithin mainly to fatty acid and 1,2diacylglycerol, indicating that it was a PLase with unique catalytic activity. PLase from V. vulnificus had temperature and pH optimum at 45°C and 7.0 in 50 mM Tris-HCl buffer, respectively, but was quite active at temperatures up to 55°C and in a broad range of pH 5 to 10. The activity of the enzyme was enhanced by divalent cations such as Ca 2+ , Co 2+ , Mg 2+ , and Mn 2+ , but not by ethylenediaminetetraacetic acid (EDTA).
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