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Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout
Author(s) -
Liu PingChung,
Chuang WenHsiao,
Tu ChihChing,
Lee KuoKau
Publication year - 2010
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201000105
Subject(s) - aeromonas hydrophila , rainbow trout , protease , cysteine , cysteine protease , biochemistry , chemistry , toxin , affinity chromatography , iodoacetic acid , microbiology and biotechnology , chromatography , enzyme , biology , bacteria , fishery , fish <actinopterygii> , genetics
An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout ( Oncorhynchus mykiss ) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L ‐cysteine, iodoacetic acid, N ‐ethylamleimide, P‐chloromercuibenzene‐sulfonic acid and N‐α‐p‐tosyl‐1‐lysine‐chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS‐PAGE. In addition, the toxin was also completely inhibited by HgCl 2 but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl 2 . Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD 50 values of 2.87 and 0.93 μg protein g –1 fish body weight, respectively. The addition of L‐cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)