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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/43442

Title: Biochemical Characterization of a Phospholipase A2 from Photobacterium damselae subsp. piscicida
Authors: Po-Yuan Hsu;Kuo-Kau Leea;Pei-Shan Lee;Chih-Chuang Hu;Cheng-Hui Lin;Ping-Chung Liu
Contributors: 國立臺灣海洋大學:水產養殖學系
Keywords: Photobacterium damselae subsp. piscicida;Rachycentron canadum;Phospholipase A2
Date: 2013-10-22
Issue Date: 2017-08-10T01:41:04Z
Publisher: Zeitschrift Fur Naturforschung Section C-A Journal Of Biosciences
Abstract: Abstract:Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of fish photobacteriosis (pasteurellosis) in cultured cobia (Rachycentron canadum) in Taiwan. A component was purified from the extracellular products (ECP) of the bacterium strain 9205 by fast protein liquid chromatography (FPLC) and identified as a phospholipase. An N-terminal sequence of 10 amino acid residues, QDQPNLDPGK, was determined by mass spectroscopy (MS) and found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. The corresponding gene sequence of the phospholipase (GenBank accession no. AB071137) was employed to design primers for amplification of the sequence by the polymerase chain reaction (PCR). The PCR products were transformed into Escherichia coli, and a recombinant protein product was obtained which was purifi ed as a His-tag fusion protein by Ni-metal affinity chromatography. A single 43-kDa band was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphatidylcholine was degraded by this protein to lysophosphatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identification of the protein as a phospholipase A2. The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 °C. The activity was inhibited by Zn2+ and Cu2+, activated by Ca2+ and Mg2+, and completely inactivated by dexamethasone and p-bromophenacyl bromide. A rabbit antiserum against the recombinant protein neutralized the phospholipase A2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD50 value between 2 and 4 μg protein/g fish. The results revealed phospholipase A2 as a fi sh toxin in the ECP of Phdp strain 9205.
Relation: 68, 471-481
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/43442
Appears in Collections:[水產養殖學系] 期刊論文

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