文献类型： 外文期刊

作者： Zhao, ML ¹ ; Huang, JS ² ; Mo, MH ² ; Zhang, KQ ² ;

作者机构： 1.Yunnan Univ, Key Lab Conservat & Utilizat Bioresource, Kunming 650091, Peoples R China

2.Yunnan Univ, Key Lab Conservat & Utilizat Bioresource, Kunming 650091, Peoples R China; Tsing Hua Univ, Dept Biol Sci & Biotechnol, Beijing 100084, Peoples R China; Chinese Acad Trop Agr Sci, State Key Lab Biotechnol & Trop Crops, Environm & Plant Protect Inst, Hainan 571101, Peoples R China

关键词： Clonostachys rosea;functional analysis;nematotoxic activity;serine-like protease

期刊名称：FUNGAL DIVERSITY （ 影响因子：20.372； 五年影响因子：16.344 ）

ISSN： 1560-2745

年卷期： 2005 年 19 卷

页码：

收录情况： SCI

摘要： A serine-like protease (designated as Lmzl) was purified to homogeneity from Clonostachys rosea. It showed a molecular mass of approximately 33 kDa, pI 10.5, optimal activity of Lmzl at 60 degrees C and pH 11-12, and broad pH stability between pH 5-12. Lmzl has a Km value for Suc-(Ala)2-Pro-Phe-pNA of 1.45 mM. The protease activity was completely inhibited by PMSF or streptomyces subtilisin inhibitor, and partially inhibited by collagenase inhibitor 1; all indicated the presence of a serine residue in the active site and Lmzl is thus most likely a member of the serine protease family. Its N-terminal amino acid sequence was directly sequenced by Edman degradation. The recombinant plasmid harbouring a cDNA gene encoding the mature protease was integrated into yeast chromosome DNA and the gene was successfully expressed as demonstrated via activity analysis, ELISA and Western blotting. The nematotoxic activity of the protease involving in fungal infection was characterised by nematode-immobilisation and cuticle degradation with the purified Lmzl or recombinant protease, and further verified by immunodepletion using anti-Lmzl antiserum prepared in mice. These observations suggest a research model for fungal infection mechanism of serine-like protease involvement.