Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum
2014
Hsu, Po‐Yuan | Lee, Kuo‐Kau | Hu, Chih‐Chuang | Liu, Ping‐Chung
Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native‐PAGE (by 0.3% egg yolk agar‐overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine ‐Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4–40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine‐tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca²⁺ and Mg²⁺ and inactivated by Zn²⁺ and Cu²⁺. Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD₅₀ values of 3.25 and 0.91 µg protein g⁻¹ fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium.
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