粘帚霉GR-6菌株种类鉴定及其β-1,3-葡聚糖酶纯化与酶学性质

Identification of Gliocladium sp. GR-6 and purification and characterization of the β-1,3-glucanase produced by GR-6

为明确粘帚霉GR-6产生的β-1,3-葡聚糖酶对植物病原真菌的作用机制,建立β-1,3-葡聚糖酶分离纯化体系,根据形态特征和ITS序列分析鉴定GR-6种类,用60%饱和度的丙酮沉淀酶蛋白质,再通过DE-52离子交换层析和Sephadex G-75凝胶柱层析纯化蛋白质。结果表明,GR-6菌株ITS序列与粉红粘帚霉的相似度最高,达到98%,结合形态学特征将该菌株鉴定为粉红粘帚霉。SDS-PAGE显示纯化后得到一种分子量为4.11×104的蛋白质,该蛋白质具有β-1,3-葡聚糖酶活性。经该酶处理后的水稻纹枯病菌(Rhizoctonia solani)和棉花黄萎病菌(Verticilliun dahliae)菌丝颜色变浅、细胞轮廓变模糊、甚至菌丝细胞逐渐消失,可见,粉红粘帚霉GR-6产生的β-1,3-葡聚糖酶对这两种植物病原真菌菌丝有直接破坏作用。纯化后的β-1,3-葡聚糖酶的最适反应温度为50℃,最适反应pH值为4,酶液在50℃以下或pH 5时保存0.5 h,酶活性基本保持稳定;该葡聚糖酶对一些金属离子较敏感,其中Zn2+、Sn2+、Ca2+、Fe2+等使酶活性显著降低,而Mn2+和K+对酶活性没有明显的影响。建立的β-1,3-葡聚糖酶纯化体系稳定可靠。

In order to make clear the mechanism of β-1, 3-glucanase from strain GR-6 of Gliocladium against plant fungal pathogens and establish the purification system of β-1, 3-glucanase, strain GR-6 was identified according to ITS sequence analysis and the morphological characteristics. The protein of GR-6 could be precipitated with 60% acetone, and purified with DE-52 and Sephadex G-75 chromatography. The results indicated that strain GR-6 shared 98% similarities with the members in Gliocladium. Combined with the morphological characteristics, the strain GR-6 was identified as Gliocladium roseum. SDS-PAGE demonstrated that the molecular weight of the purified protein was about 4. 11×10^4 and had β-1, 3-glucanase activity. When the mycelia of Rhizoctonia. solani and Verticillium. dahliae were treated by the glucanaseprotein, it was observed that the outline of hyphal cells became illegibility, the color of hypha became depigmentation, and even some hyphae presented abnormality or collapsed completely, which proves the β-1, 3-glucanase can destroy mycelia of the two plant fungal pathogens. The optimal temperature for the hydrolyzation of the glucanase was 50 ℃and the optimal pH value was 4.The glucanase activity was stable when it was heated for 0. 5 h at 50 ℃and kept in the solution with pH 5. The enzymatic protein was sensitive to some ions; it was inhibited by Zn2 ＋ , Sn2 ＋ , Ca2 ＋ , Fe2 ＋ and was not affected by Mn2 ＋ and K＋ . A reliable purification system of β-1, 3-glucanase was developed.