鱼源溶藻弧菌胞外产物的特性研究
CHARACTERIZATION OF EXTRACELLULAR PRODUCTS FROM VIBRIO ALGINOLYTICUS ISOLATED FROM MARICULTURED FISH
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摘要: 溶藻弧菌经胰胨大豆胨琼脂培养基(TSA+2%NaCl)培养48h后,培养物用PBS洗脱、离心,再经孔径0.22μm微孔滤膜过滤,制得胞外产物(Extracellular product,ECP)。经检测,该ECP具有淀粉酶、酪蛋白酶、卵磷脂酶和脂肪酶等4种酶活性,其中淀粉酶的活性最高,酪蛋白酶、脂肪酶活性次之,卵磷脂酶活性较低,未检测出明胶酶及脲酶活性;能溶解断斑石鲈(Pomadasys hasta)、眼斑拟石首鱼(Sciaenops ocellatus)和赤点石斑鱼(Epinephelus akaara)的红细胞,而不能溶解鸡、羊及O型人红细胞;对小白鼠和石斑鱼的LD50分别为0.08mg/g和0.17mg/g,95%可信限分别为0.06-0.12mg/g和0.07-0.28mg/g;50mmol/L EDTA和100mmol/L苯甲基磺酰氟能使胞外蛋白酶(Extracellular pro-teinase,ECPase)的活性分别下降83.40%和51.32%;ECPase对热稳定性较差,碱性条件下活性较高,其最适温度为50℃,最适pH为8.0。Abstract: Vibrio alginolyticus is an opportunist pathogen，which exists extensively in seawater，and causes diarrhea in humanand vibriosis in maricultured animals. Fewer studies on the extracellular product(ECP) of V. alginolyticus were reported. In our experiments the ECP was extracted from V. alginolyticus and the biological and physicochemical characters of the ECP were as-sessed. After V. alginolyticus had grown on tryptic soy agar(TSA) supplemented with 2％NaCl for 48h，the bacteria on the Petri dish were washed away by PBS，and the bacteria suspension was centrifuged at 10000 r/ min at 4 0C, then the supernatant was filtered through 0. 22μmfilter to obtain the extracellular products(ECP).The results showed that the ECP of V. alginolytics has amylase，casease，lecithinase and lipase activities，which the amylase activity was highest，casease and lipase activities sec-ondly，and lecithinase lowest，but has no gelatinase and urase activities. The ECP could dissolve the erythrocytes of lined silver grouper(Pomadasys hasta)，red drum(Sciaenops ocellatus) and redspotted grouper(Epinephelus akaara)，but not the ery-throcytes of chicken，sheep and o-type blood human. The LD50 value of ECP on mice and E. akaara were 0. 08 mg/ g and 0. 17mg/ g respectively，the 95%credibility ranges were 0. 06 -12mg/ g and 0. 07-28mg/ g respectively. 50mmol/ L ethylene-diamine tetra-acetie acid(FDTA) and 100mmol/L phenylmethylsulfonyl fluoride(PMSF) could reduce extracellular proteinase(ECPase) activities of V. alginolyticus by 83. 40%and 51.32%separately. The ECPase activities gradually rose with the in-crease of temperature from 4 ℃ to 50℃，and highest at 50 ℃，then dropped suddenly. The ECPase activities were gradually raided also with the enhancement of pH value，and highest at pH8.0，moreover higher at pH10. 6. Thus the ECPase has bad ther-monstability，and the optimal temperature and pH value of the ECPase is 50℃ and pH8.0 respectively.
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Keywords:
- Vibrio alginolyticus /
- Extracellular product /
- Character
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