Purification, Properties And Antifungal Activity Of Endo-β-1, 3-Glucannase Produced By Talaromyces Flavus

Mycoparasites are an important group of biocontrol fungi. The mycoparasitic fungus Talaromyces flavus parasitizes S. sclerotiorum, Sclerotium rolfsii, and Rhizoctonia solani. Cell wall-degrading enzymes, such as beta-1,3-glucanase, cellulase, and chitinase, are involved in the mycoparasitism. The biological characteristics of Talaromyces flavus and mycoparasitism have been studied. However, the purification, properties of the beta-1,3-glucanase have not been reported yet in the world. Studies suggest that a rich source of cell wall-degrading enzymes from T. flavus that can be used to control diseases in plants.Our studies show that the mycoparasitic fungus T. flavus produces at least three extracellular cell wall-degrading enzymes, chitinase, cellulase and beta-1,3-glucanases in induced culture. The optimal culture for producing beta-1,3-glucanases is SMF (Synthetic Medium with Fungal cell walls as carbon sources) culture, which contains 0.1g(NH4)2SO4, 0.3g MgSO4·7H2O, 0.4g KH2PO4, 0.8g K2HPO4, 0.4g KNO3, 0.2g CaCl2, 2mg ZnSO4, 2g yeast extract, fungal mycelia cell walls(10g fungal mycelia, dry weight) per 1000ml。This study focused on the purification, properties and antifungal activities of endo beta-1,3-glucanase from T. flavus. T. flavus was cultured in SMF at 28℃ on a rotary shaker (200rpm) for 7 days. An endo-beta-1,3-glucanase (EC3.2.1.39) was purified from T. flavus by fractional precipitation with ammonium sulphate, ion-exchange chromatography (DEAE-Sepharose Fast Flow), Phenyl-Sepharose Fast Flow chromatography and gel-filtration chromatography (Sephacryl S-100). Determined by 12% SDS-PAGE and gel filtration, the molecular weight of beta-1,3-glucanase was about 41.0~42.6kDa. Periodec Acid-Schiff staining shows that this enzyme was a glycoprotein. Using a substrate specificity analysis, it was shown that the endo-beta-1,3-glucanase hydrolysed exclusively linear 1,3-beta-glucan chains. The endo-beta-1,3-glucanase had an optimum pH of 4.0~4.5 and an optimumtemperature of 40 degrees C. By Michaelis-Mente's kinetics assay, the Km and Vmax of The endo-beta-1,3-glucanase were 1.74 mg/mL, 0.176 mg/mL·min, respectively. Different metal ions showed different effects on the endo-beta-1,3-glucanase activity. Na2+,Ca2+,Mn2+ enhanced activity, whereas Hg2+,Cu2+,Ag+ caused obvious inhibition.The endo-beta-1,3-glucanase showed antifungal activities against tested fungi and more effective against fungi of Sclerotinia sclerotiorum, Alternaria longipes and Rhizoctonia solani .