Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium.
Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium.
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DOI：
10.1139/m90-093
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发表时间：
1990-08
影响因子：
2.8
通讯作者：
L. V. Lopez-Llorca
中科院分区：
文献类型：
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作者：
L. V. Lopez-Llorca
孢囊线虫卵的真菌寄生在以明胶为唯一氮源和碳源的半液体培养中，产生胞外蛋白水解酶。培养滤液的蛋白分解活性在接种后12-14天达到最大值。Sephadex G-100凝胶过滤层析分离出两个蛋白水解峰。经SP-Sephadex C-25离子交换层析进一步纯化为单峰。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，该酶的相对分子质量为32 kDa。该酶是一种降解酶谱中纤维蛋白原的内肽酶，以异硫氰酸荧光素-酪蛋白为底物，最适pH为8.5。苯甲基磺酰氟对该酶有抑制作用，表明该酶为丝氨酸蛋白酶。纯化的蛋白水解酶能够降解某些孢囊线虫蛋白，提示32 kDa蛋白的参与和特异性。
The fungal parasite of eggs of cyst nematodes, Verticillium suchlasporium, produced extracellular proteases when grown in semiliquid culture with gelatin as the only source of nitrogen and carbon. The proteolytic activity of culture filtrates was maximum 12–14 days after inoculation. Gel filtration chromatography in Sephadex G-100 resolved two peaks of proteolytic activity. The peak accounting for most of the activity was further purified by ion-exchange chromatography in SP-Sephadex C-25 as a single peak. This protease had a molecular mass of 32 kDa calculated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The enzyme was an endopeptidase that degraded fibrinogen in zymograms and had an optimum pH of 8.5 using fluorescein isothiocyanate – casein as the substrate. It was inhibited by phenylmethylsulfonyl fluoride, indicating that it was a serine protease. The purified protease was able to degrade certain cyst nematode proteins suggesting the involvement and specificity of the 32-kDa protea...