Lee K K, Yu S R, Liu P C
Department of Aquaculture, National Taiwan Ocean University, 2, Pei-Ning Road, 20224, Keelung, Taiwan, R.O.C.
Curr Microbiol. 1997 Feb;34(2):110-7. doi: 10.1007/s002849900153.
An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn(Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose Hig hPerformance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),and showed maximal activity at pH 10, having a molecular weight of about 33kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl2 but partially inhibited by CaCl2, CuCl2, CoCl2,MnCl2, and ZnCl2, and not inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecylsulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD50 values of 0.30 and 0.27 microg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy.
最初从患病的日本对虾（Penaeus japonicus）中分离出的溶藻弧菌Swy菌株产生的一种细胞外致死毒素，通过具有疏水相互作用（苯基琼脂糖高性能）色谱和凝胶过滤柱的快速蛋白质液相色谱进行了部分纯化。该毒素是一种碱性丝氨酸蛋白酶，被苯甲基磺酰氟（PMSF）抑制，在pH 10时表现出最大活性，通过SDS-PAGE和凝胶过滤色谱估计其分子量约为33kDa。此外，该毒素也被FeCl2完全抑制，但被CaCl2、CuCl2、CoCl2、MnCl2和ZnCl2部分抑制，且不被乙二胺四乙酸（EDTA）、乙二醇双（β-氨基乙醚）N,N,N',N'-四乙酸（EGTA）、碘乙酰胺、胃蛋白酶抑制剂A、十二烷基硫酸钠（SDS）和N-对甲苯磺酰基-L-苯丙氨酸氯甲基酮（TPCK）抑制。粗细胞外产物（ECP）和部分纯化的毒素对日本对虾均具有致死性，其LD50值分别为0.30和0.27微克蛋白质/克体重。添加PMSF完全抑制了ECP和部分纯化毒素的致死毒性，表明这种丝氨酸蛋白酶是该细菌产生的致死因子。因此，推测33kDa蛋白酶是溶藻弧菌Swy菌株产生的一种新的有毒蛋白酶。
