碩士 / 國立海洋大學 / 水產養殖學系 / 89 / This study investigated the pathogensis of Vibrio carchariae EmI82KL strain, isolated from the intestinal yellow fluid of grouper ( Epinephelus coioides ) ,in the grouper ( Epinephelus sp.). Strains Rd (isolated from red drum Sciaenops ocellatus ) and SfUSA (isolated from summer flounder Paralichthys dentatus ) were also used in this study for comparison.
An apparently lower protease activities in the extracellular products ( ECP ) of strains EmI82KL、Rd and SfUSA were obtained when the bacteria grew on tryptone soya agar ( TSA, supplemented with 1.5 % NaCl ) plates compared to those grew on 1.5 % eel meal agar ( +1.5 % NaCl) or modified seawater complete agar ( MSWCA ). The protease activity of ECP hide power azure digestibility from strains EmI82KL、Rd and SfUSA were 5325、3665 and 3877 units/mg protein, respectively. The protease activity in the ECP of all the three strains could be inhibited by Phenylmethanesulfonyl fluoride ( PMSF )、N-*-p-tosyl-l-lysine-chloromethyl ketone ( TLCK ) and N-tosyl-l-phenylalanine-chloromethylketone ( TPCK ) indicating that it is a serine protease.
In toxicity test, all the groupers ( 4.4g ) were killed after intraperitoneal (i.p.) injection of the EmI82KL strain with a dose of 108 colony forming units ( CFU )/g fish body weight, and the LD50 of Rd strain for red drum was 2.9 × 107 CFU/g fish body weight. The LD50 of ECP of EmI82KL、Rd and SfUSA strains for the groupers were 6.841、8.872 and 5.788 while for red drum were 5.994、2.798 and 4.604 *g protein / g fish body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of the ECP of each strain tested.
A 33 KDa (determined on SDS-PAGE) protease was purified from ECP of EmI82KL strain grew on MSWCA plate by using various columns (hydrophobic interaction chromatography column, RESOURCE Q column and Mono Q column ) on Fast Protein Liquid Chromatography system. The purified protease was completely inhibited by serine protease inhibitors — PMSF、TLCK and TPCK, and partially inhibited by cysteine protease inhibitors — L-cysteine and partially inhibited by metallo protease inhibitor —ethylenediaminetetraacetic acid (EDTA)、ethylene glyco-bis(B-amino-ethyl ether) N,N,N’,N’-tetraacetic acid (EGTA). The optimum pH of the protease was 6 — 8 and it was heat labile.
| Identifer | oai:union.ndltd.org:TW/089NTOU0086021 |
| Date | January 2001 |
| Creators | Wen Hsiao Chuang, 莊文 |
| Contributors | Lee Kuo Kau, 李國誥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 54 |
Page generated in 0.0015 seconds