螺旋毛壳ND35 β-1,3-葡聚糖酶的诱导、性质及其抑菌作用  

Induction and characterization of β-1,3-glucanases from the mycoparasite Chaetomium spirale

作　　者：郭晓[1] 高克祥[1,2] 印敬明[1] 白复芹[1] 马迎新[1] 于丹[1] 刘晓光[1] 

机构地区：[1]山东农业大学植物病理系,泰安271018 [2]西北农林科技大学植物保护学院

出　　处：《植物病理学报》2005年第6期493-503,共11页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(30100143)

摘　　要：以病原菌Rhizoctonia solani的细胞壁为诱导物,模拟毛壳菌自然的重寄生过程,研究了内生真菌螺旋毛壳(Chaetomium spirale)ND35 β-1,3-葡聚糖酶的产酶条件、性质,尤其是不同碳源的调控作用。结果表明,不同种类的真菌细胞壁及几丁质 和昆布多糖,均可诱导产生β-1,3-葡聚糖酶,而作为分解代谢产物的葡萄糖则抑制产酶。经硫酸铵沉淀、DEAE-Sepharose 阴离子交换层析及Phenyl-Sepharose疏水层析,并通过SDS-PAGE鉴定,纯化了一种分子量约为73 kDa的内切β-1,3-葡聚 糖酶GLUC73。其最适反应温度为55℃,在40℃以下较稳定;最适pH值为5．5,在pH 5-9范围内均很稳定;酶活性受 Hg2+、Fe3+、Zn2+、Mg2+等金属离子不同程度的抑制,Mn2+和Co2+对酶有激活作用;以昆布多糖为底物时,该酶的米氏常数 Km为0．412 mg·mL-1,最大反直速度Vmax为3．876 U·mL-1。粗酶液同时具有β-1,3-葡聚糖酶和几丁质酶活性,离体抑 菌试验表明,对苹果炭疽病菌(Glomerella cingulata)、杨树腐烂病菌(Valsa sordida)、苹果树腐烂病菌(Valsa mali)的菌丝 生长和孢子萌发有明显的抑制作用。通过对β-1,3-葡聚糖进行免疫细胞化学标记和超微结构观察,间接证明了β-1,3-葡 聚糖酶在螺旋毛壳重寄生过程中的作用。Induction,properties and antifungal activity of β-1,3-glucanases from endophytic fungus Chaetomium spirale ND35 were reported in this study. The production of β-1,3-glucanases was influenced by the carbon sources used. In synthetic medium （ SM ）, cell wall preparation （CWP） of several fungi, including Valsa sordida, Saccharomyces cerevisiae, Rhizoctonia cerealis and R. solani, as well as polysaccharides such as colloidal chitin and laminarin could induce β-1,3-glucanases in C. spirale ND35. However, high level of glucose as catabolite repressed the production of β-1,3-glucanases. An endo-β-1,3-glucanase GLUC73 was purified to electrophoretic homogeneity through ammonium sulfate precipitation and DEAE Sepharose Fast Flow anion-exchange chromatography, followed by Phenyl Sepharose Fast Flow hydrophobic chromatography. Its molecular mass is about 73 kDa by SDS-PAGE. It functioned optimally at 55℃ and pH 5.5 and was stable over a broad range of pH 5 -9 and below 40℃. The endo-β-1,3-glucanase activity was inhibited by Hg^2＋, Fe^3＋ ,Zn^2＋ and Mg^2＋ to different extents and stimulated by Mn^2＋ and Co^2＋. The Km and WmaxValUeS for the β-1, 3-glucanase,using laminarin as substrate, were 0. 412 mg · mL^-1 and 3. 876 U · mL^-1, respectively. In addition,the bioassay of antifungal activity in vitro showed that the crude extract with both β-1,3-glucanase and chitinase activities, significantly suppressed mycelial growth and conidia germination of several phytopatho- genic fungi such as Glomerella cingulata, V. sordida and V. mali. Further TEM ultrastructural investigation on interaction between C. spirale ND35 and R. solani by immuno-cytochemical labeling provided indirect proof that β-1,3-glucanase played a key role in mycoparasitism.

关 键 词：螺旋毛壳菌ND35 β-1 3-葡聚糖酶 酶学性质 抑菌作用 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]