Extraction, Purification And Characterization Of Grifola Frondosa β-glucan Synthase

Taking Grifola frondosa strain Gf-3as material, Grifola frondosa β-glucan synthase（GFβGS） was extracted and purified from the fermentation mycelium of submergedfermentation, and its enzymatic properties were studied.The mycelium was broken by ultrasonic, centrifugated and the supernatant was collected.GFβGS was precipitated by different concentrations of acetone, ammonium sulfate andethanol, the suitable isolation method of GS was researched out by taking enzyme activity andprotein content as parameters. After24hours on standing, centrifugated and the pellets werecollected.GFβGS crude enzyme was purified by recrystallization twice and native-PAGE,electrophoretically pure GFβGS can be obtained.The molecular weight of GFβGS is55057.09Da. The optimum pH of GFβGS enzymereaction is pH=5.0and the optimum reaction temperature is15°C, GFβGS is most stable atpH=5.0and in the range of30°C to50°C.Ca²⁺, Mg²⁺and Al³⁺can activate GFβGS to a certain extent. Mn²⁺havr had significantactivation on GFβGS with maximum activation occurring at3mmol/L, the GFβGS specificactivity is2.83times as against that of the blank control group. Cu²⁺and Ba²⁺have inhibitingeffect on GFβGS to a lesser degree. Zn²⁺and Fe³⁺have obvious inhibiting action on GFβGS,4mmol/L Zn²⁺and3mmol/L Fe³⁺can reduce the GFβGS specific activity of82%and66.7%respectively. Fe²⁺has no effect on the activity of GFβGS.GFβGS is an intracellular enzyme and without grifolan synthase activity in fermentationliquor or extracellular.GFβGS is a one-way enzyme that it can only catalyze the synthesis of β-glucan. Theconsumption of substrate glucose was completely catalyzed to the synthesis of Grifolafrondosa β-glucan （Grifolan） by GFβGS, and any polysaccharide with α structure is not beenfound in the catalytic reaction products. The structure of GFβGS reaction products andGrifolan are basically the same. This study can lay the foundation for the further study on structure and function ofGFβGS, and provide the basis for the thorough study of Grifolan synthesis mechanism andimproving the Grifolan production efficiency. It can also lay the foundation for the study ofβ-glucan synthase inhibitors new antifungal antibiotic.