Wang Bin, Wu Wenping, Liu Xingzhong
Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China.
Mycopathologia. 2007 Mar;163(3):169-76. doi: 10.1007/s11046-007-0100-y. Epub 2007 Mar 15.
Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40 degrees C, respectively. The Hnsp activity was stable at pH 6-8 and decreased radically at 50 degrees C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode.
丝氨酸蛋白酶在真菌感染无脊椎动物宿主过程中发挥着重要作用。在以线虫（重生滑刃线虫）作为唯一氮源的罗西里被毛孢OWVT-1液体培养物中检测到一种细胞外蛋白酶（Hnsp），并通过硫酸铵沉淀、阴离子交换色谱和凝胶过滤将其纯化至同质。其分子量约为32 kDa，最佳反应pH值和温度分别为pH 7和40℃。Hnsp活性在pH 6 - 8时稳定，在50℃下10分钟急剧下降。Hnsp对苯甲基磺酰氟抑制剂高度敏感，且能很好地分解底物N-琥珀酰-丙氨酸-丙氨酸-脯氨酸-苯丙氨酸-对硝基苯胺，表明它属于丝氨酸蛋白酶的胰凝乳蛋白酶/枯草杆菌蛋白酶。Hnsp的N端氨基酸序列为SVTDQQGADCGLARISHRE，与其他食线虫真菌的丝氨酸蛋白酶具有高度同源性。体外杀死线虫并降解其角质层的能力表明Hnsp可能参与对线虫的感染。
