Optimization Of Aspergillus Niger β-glucosidase Fermentation Medium And The Hydrolysis Of Dioscin

Trillin is an active component in traditional Chinese medicine. It can be used as a valuable precursor for the synthesis of some other bioactive saponins. In addition, trillin is highly active against a broad-spectrum of plant fungal pathogen and thus has the potential to be developed as an effective biological fungicide. However, it is difficult to extract trillin at a large scale of trillin directly from medicinal plants. In this study, the partially purified P-glucosidase from Aspergillus niger CICC2475 was explored to hydrolyze the diosgenin saponins isolated from Dioscorea zingiberensis to trillin.Fermentation medium for β-glucosidase production from A. niger CICC2475 was optimized as:bran 20 g/L, (NH4)2SO41.5 g/L, KH2PO42 g/L, MgSO4-7H2O 0.2 g/L, CaCl2 0.01 g/L, FeSO4 0.01 g/L), the highest activity of β-glucosidase (42.7U/ml) was obtained.The extracellular 《-glucosidase was isolated and purified from fermentation broth by differential 30% and 80% saturation ammonium sulfate precipitation and Superdex 200 gel filtration chromatography. The molecular weights of the β-glucosidases were determined by SDS-PAGE to be about 115 kDa and 70 kDa, the specific enzymatic activity 62.1 U/mg and 53.0 U/mg, the recovery rate 19.6% and 19.1%, respectively.Enzymatic properties of β-glucosidase were investgated. The optimum pH and temperature for β-glucosidase were 4.6 and 50℃-55℃, respectively. The enzyme was stable in the pH value range of 4.2-4.8, and at the temperature between 40℃ to 60℃. The Km and Vmax values of the enzyme were determined as 2.33 mmol/1 and 3.14 mmol/(L-min) when pNPG was used as a substrate.The hydrolytic product of dioscin by the partially purified β-glucosidase was characterized as trilium glycosides. The optimum conditions for dioscin hydrolysis by the A. niger P-glucosidase was:100U of β-glucosidase, pH4.6,0.4% substrate dissolved into DMSO, and 100U of β-glucosidase, temperature 50℃,24 h incubation. Under such hydrolysis conditions,0.21 mg/ml of trillin was obtained.