Lee K K, Yii K C, Yang T I, Hong H I, Liu P C
Department of Aquaculture, National Taiwan Ocean University, Keelung.
Z Naturforsch C J Biosci. 1999 May-Jun;54(5-6):383-6. doi: 10.1515/znc-1999-5-613.
Protease and virulence of the extracellular products (ECP) of Vibrio carchariae strain EmI82KL, a causative agent of gastroenteritis in Epinephelus coioides, cultured on different media were studied. The bacteria grown on peptone agar, nutrient agar or brain heart infusion agar produced higher protease activities than that grown on tryptic soy agar (TSA) in terms of protein content. The addition of ethylenediamine di(o-hydroxyphenylacetic acid) or horse serum in TSA enhanced the ECP protease production while the addition of grouper serum apparently reduced the enzyme activity indicating the presence of protease inhibitor(s) in the fish serum. Furthermore, the use of grouper meat or peptone as a single nutrient source remarkably enhanced the production of ECP protease. Adding proteinaceous materials from animal sources (horse serum, grouper meat or peptone) on agar manifested higher ECP protease activity than that from plant source (TSA), indicating the intestine of carnivorous groupers might favour the existence, survival or infection of the bacterium. The protease was a metal-chelator-sensitive serine protease since it was inhibited by 3,4-dichloroisocoumarin and phenylmethanesulfonyl fluoride while about 80% of its activity inhibited by chelating agents (ethylene-diaminetetraacetic acid and ethylene glycol-bis(beta-amino-ethylether) N,N,N',N'-tetraacetic acid). The ECP obtained from each medium was not lethal to the groupers suggesting that the bacterium is low virulent. As grouper is carnivorous, therefore, the role of the protease played in the fish intestine probably is competing for nutrients and/or associated with the cause of edema leading to gastroenteritis.
研究了斜带石斑鱼肠胃炎病原体——鲨弧菌菌株EmI82KL在不同培养基上培养时细胞外产物（ECP）的蛋白酶活性和毒力。就蛋白质含量而言，在蛋白胨琼脂、营养琼脂或脑心浸液琼脂上生长的细菌所产生的蛋白酶活性高于在胰蛋白胨大豆琼脂（TSA）上生长的细菌。在TSA中添加乙二胺二（邻羟基苯乙酸）或马血清可增强ECP蛋白酶的产生，而添加石斑鱼血清则明显降低酶活性，这表明鱼血清中存在蛋白酶抑制剂。此外，使用石斑鱼肉或蛋白胨作为单一营养源可显著提高ECP蛋白酶的产量。在琼脂上添加动物源蛋白质材料（马血清、石斑鱼肉或蛋白胨）所表现出的ECP蛋白酶活性高于植物源材料（TSA），这表明肉食性石斑鱼的肠道可能有利于该细菌的生存、存活或感染。该蛋白酶是一种对金属螯合剂敏感的丝氨酸蛋白酶，因为它可被3,4-二氯异香豆素和苯甲基磺酰氟抑制，同时约80%的活性可被螯合剂（乙二胺四乙酸和乙二醇双（β-氨基乙醚）N,N,N',N'-四乙酸）抑制。从每种培养基中获得的ECP对石斑鱼无致死性，这表明该细菌的毒力较低。由于石斑鱼是肉食性的，因此，蛋白酶在鱼肠道中所起的作用可能是争夺营养物质和/或与导致肠胃炎的水肿原因有关。
