The Mutation Of β-1,3-glucanase By GR,Purification, Characterization Of The β-1,3-glucanase And Identification Of GR Strain

GR was a fungus isolate, which could inhibit a variety of pathogens. The GR was identified as Gliocladium roseum by the colony morphology, conidiophore structure, spore morphology and ITS DNA sequence analysis.GR could secreteβ-1,3-glucanase. The inhibition experiment of the enzyme on plant pathogens showed that the crude enzyme significantly inhibited the hyphal growth of Rhizoctonia solani and Verticillium dahliae. In addition, It was observed that the some hyphal color shallow, cell swelling, protoplast concentration, severe hyphal cell rupture when Rhizoctonia solani mycelium were treated with theβ-1,3-glucanase, while some Verticillium dahliae top hypha cell became vesicle and some hyphal cells were destroied which led to protoplasm leakage from the wound after the pathogen mycelium treament by the enzyme.High-yieldβ-1,3-glucanase mutant GR-6 was screened by UV inducing from GR isolate. Theβ-1,3-glucanase activity of GR-6 was 2.8 times comparatively with it's parent isolate. It had better genetic stability that GR-6 metabolizedβ-1,3-glucanase. The characterizations of GR-6 were approximately the same as that of the parent strain, such as hyphal growth rate and asexual ability.Theβ-1,3-glucanase of GR-6 could be precipitated by 60% acetone. The crude enzyme could be obtained with suspending that precipitate in buffer. Theβ-1,3-glucanase protein was purificated by chromatography. The crude enzyme protein displayed 5 absorption peaks at 280nm, and peak 4 protein possessedβ-1,3-glucanase activity using DE52 hydrophobic chromatography. The peak 4 protein was collected and purificated by Sephadex G-75 gel filtration, the protein of peak 4 was separated into 3 protein peaks, which the 4-1 with activity was collected as the protein ofβ-1,3-glucanase. The molecular weight of the enzyme protein was determined by SDS-PAGE to be about 41.1kDa.Characterizations of theβ-1,3-glucanase were detected in this paper. The optimum temperature for the enzyme activity was 50℃and the range of the optimum pH was between 4-5. Theβ-1,3-glucanase activity was stable when that was treated for 30 min by hot water under 50℃, and the activity was distinct declining at 60℃persisting for 30min.. The enzyme activity was stable when the enzyme protein was in the solution with pH 5, while the activity decline markedly in the solution with pH 4 and pH6.The some metal ions could inhibit theβ-1,3-glucanase activity when the enzyme solution amended with metal ions respectively, such as Ca2+,Zn2+,Fe2+,Fe3,Ba2+,while others no evident effect, such as Mn2＋,K＋.It were optimized that the culture conditions for GR-6 isolate produced theβ-1,3-glucanase. The analysis results indicated that appropriate carbon nutrition was glucan and the concentration was 2%, nitrogen nutritions was ammonium nitrate and the concentration was 0.7%, the medium primary pH value was 5, the culture temperature was 26℃and the time of incubation GR-6 was 14d.