Purification And Partial Properties Of β-1,3-glucanases Produced By Gaeumannomyces Graminis Var. Tritici

Take-all is one of the destructive diseases in wheat which widely distributed in China and other countries. Because of the absence'of resistant varieties and effective chemicals, as well as the well understanding of pathogenesis, the disease still seriously affects the production and quality of the wheat.Gaeumannomyces graminis var. tritici, the cause of take-all disease, secrets not only cellulase, xylanase, but also β-1,3- glucanases which decomposes (3-glucans in penetrating lignituber for its extension and colonization in the process of infection. The producing conditions of extracellular β-1,3- glucanases by Gaeumannomyces graminis var. tritici and properties of crude enzyme were reported by our research group. In this study β -1,3-glucanases produced by Gaeumannomyces graminis var. tritici were purified and the properties of pure enzyme were studied. The results will help to understand the mechanisms of cytochemistry, molecular of pathogenicity and biological genetic engineering of β-1,3- glucanases.The results are as follows:1 , After native polyacrylamide gel electrophoresis(the polyacrylamide resolving gel 7.5%, the stacking gel 3.9%), the two isozyme produced by Gaeumannomyces graminis were visualized by activity-staining, coomassie Brilliant Blue R-250 staining and silver staining.2, p-l,3-glucanase I and p-l,3-glucanasellby the steps of 90% ammonium sulfate precipitation, Sephadex G-100 and DEAE Sephadex A-50 anion interaction column chromatograph were obtained.3, Accroding to the standard high molecular mass marker, the molecular mass of thep-l,3-glucanase I and p-l,3-glucanasellwerel20 kilodaltons and 150 kilodaltons respectively by discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (the polyacrylamide resolving gel 7.5%, the stacking gel 3.9%).4. The study of purified enzyme properties showed that the optimal temperature and pH of p-l,3-glucanase I were 60℃ and pH 4.5, this enzyme was stable at pH 5.0 ~ 8.0 and temperature<40℃ ; the optimal temperature and pH of p-l,3-glucanasellwere 30℃ and pH 7.5, this enzyme was stable at pH 7.5 and temperature <20℃. 5, The pi of β-1,3-glucanase I was 3.9 by isoelectric focusing.The two isozyme produced by Gaeumannomyces graminis were firstly proved byactivity staining, coomassie Brilliant Blue R-250 staining and silver staining. At the same time, p-l,3-glucanase I and p-l,3-glucanasellby the steps of ammonium sulfate precipitation, Sephadex G-100 and DEAE Sephadex A-50 anion interaction column chromatograph were obtained. It provided the science basis for further study of the pathogen penetration and extension, the relationship between p-l,3-glucanases and pathogenisis.