title: Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum.
abstract: 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 Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . 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.
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Po-Yuan
authors.initials: PY
authors.lastname: Hsu
authors.forename: Kuo-Kau
authors.initials: KK
authors.lastname: Lee
authors.forename: Chih-Chuang
authors.initials: CC
authors.lastname: Hu
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Liu
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D005399:Fishes / Q000382:microbiology*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007928:Lethal Dose 50; D008970:Molecular Weight; D010776:Photobacterium / Q000201:enzymology*; D013696:Temperature; D010738:Type C Phospholipases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D006460:Hemolysin Proteins; D010738:Type C Phospholipases
keywords: Cobia; Extracellular products; Fast protein liyuid chromatography; Phospholipase; Photobacterium damselae subsp. piscicida; Rachycentron canadum