Screening Of B-Glucosidase Producing Strains And Research On Its Immobilization, Isolation And Purification

β-glucosidase, a key component of cellulose, has been widely applied to food, medicines, chemical industries and other aspects. This research begins to screen from high actively β-glucosidase producing strains for Aspergillus niger M97. Then it aims to elevate the activity of producing enzymes by optimizing the fermental enzyme production conditions of Aspergillus niger M97. Also the optimized β-glucosidase will be immobilized to analyze its enzymatic properties. At the same time, electrophoretically pure β-glucosidase is separated from the fermentation liquid.Main research conclusions:1. Aspergillus niger M97 is screened as the producing strains of P-glucosidase. Through single-factor and response analysis experiments, optimization is conducted on the culture conditions that Aspergillus niger M97 produces P-glucosidase. The optimum culture condition is as follows.(1) Composition of the fermentation culture medium:wheat bran and corncob lOg (63:37), solid-to-liquid ratio 1:1.5, nutrient solution includes (NH4)2SO4 2.0%、 NaCl 0.3%、MgSO4 0.1%、KH2PO4 0.2%、Tween80 0.2%.(2) Fermental cultivation conditions:lOmL inoculum size with microsporum canis suspension (108/mL),9-layer-gauze sealing, pH unchanged,5days, temperature 30℃.The activity of Aspergillus niger M97 producing β-glucosidase is respectively 1.22 times and 2.99 times than that of initial one after optimization on the composi-tions and the conditions.2. With chitosan,sodium alginate or their composite as base material, β-glucosi-dase can effectively be immobilized, and the immobilization ratio is respectively 85.38%,69.36% and 82.60%; and the enzyme activity is respectively 6.98IU/g wet gel、 2.80IU/g wet gel、6.07IU/g wet gel。The properties of these three immobilized enzymes are as follows:(1) The optimum pH, pH stability, the optimum temperature and temperature stability of immobilized β-glucosidase have different degrees of improvement, compared with free enzyme. Among them, the chitosan immobilized enzymes show obvious alkali resistance and high temperature resistant.(2) All the Michaelis constants of the three immobilized enzymes are lower than that of the free enzyme. It signifies that immobilized enzymes have better affinity for substrates than free enzyme, which is beneficial to the reaction.(3) As the reaction is to occur in the 50 ℃ water bath, only the chitosan immobi-lized enzyme will stay as complete structure, therefore, the operational stability analy-sis is to solely conduct on it. After 10 recycling use, the activity of residual enzyme is still 62% of the original. It illustrates that the chitosan immobilized enzyme poss-esses fine stability.(4) Store immobilized enzymes and free enzyme in the buffer solution of citric acid and disodium hydrogen phosphate, with temperature 4℃ and pH 4.8, the acti-vity of free enzyme reduces to 50% in 25 days, while the immobilized enzymes keep over 50% in a month, which proves that the storage stability of the latter is obviously higher than the former.3. Through ammonium sulfate precipitation, dialysis desalination and DEAE52 ion chromatography, electrophoretically pure β-glucosidase is obtained from the crude β-glucosidase. Its purification fold is 5.17 and the yield 15.62%. The purified β-glu -cosidase is of molecular weight 95KDa, optimum pH4.0, relatively stable in pH3.0-6.0, optimum temperature 60℃, relatively stable in 40-65℃ and Michaelis constant 1.03mmol/L.