Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lee) of V cholerae. The recombinant protein (rPhlA) produced from the phlA gene of V. mimicus was expressed in Escherichia coli as His-tag fused protein. The rPhlA was purified by gel filtration and Ni-metal affinity chromatographies. When the action mode was investigated by TLC and GC-MS, the purified rPhlA protein showed a phospholipase A activity, which cleaved the fatty acids at the sn-1 and sn-2 positions of phosphatidylcholine. However, it did not show lysophospholipase, sphingomyelinase, and phospholipase C activities. The rPhlA showed maximum activity at temperature of about 40degreesC and pH around 8-9. Some divalent cations could affect the activity of PhlA. The addition of Co2+ increased the activity, whereas Mg2+ and Zn2+ did not enhance the enzyme activity. The rPhlA could lyse the erythrocytes obtained from the fish such as rainbow trout and tilapia. A significant cytotoxic activity on a fish cell line, CHSE-214, was observed after 24 h exposure to 40 mug rPhlA protein. (C) 2002 Elsevier Science (USA). All rights reserved.