螺旋毛壳ND35 β-1,3-葡聚糖酶的诱导、性质及其抑菌作用

 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K_m为0.412 mg·mL^-1,最大反直速度V_max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 and characterization of β-1,3-glucanases from the mycoparasite Chaetomium spirale

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 Hg²⁺, Fe³⁺,Zn²⁺and Mg²⁺ to different extents and stimulated by Mn²⁺and Co²⁺. The K_m and V_max values 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 phytopathogenic fungi such as Glotnerella 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.