Study On Breeding Of β-glucanase Producing Strain, Fermentation And Properties Of β-glucanase

β-glucanase is an important industrial enzyme,it could degrade theβ-glucan in corn such as barly, so it's especially important in brewing and feed stuff industries.The breeding ofβ-glucanase high-producing strain, the fermentation condition and medium of flask fermentation and character ofβ-glucanase were investigated in this thesis. The main results were as follows:By UV radiation mutation method to the parental strain Aspergillus usamii M12, M12-9-5, aβ-glucanase producer was derived with enzyme activity of 57.85U/mL in the flask fermentation, which was increased by 88% compared with the parent stratin after the single colony separation, plate screen and flask fermentation experiment. The new mutant strain was stable for theβ-glucanase activity production. The condition of flask fermention was determined through single factor experiments: temperature 30℃, initial pH 5.5, medium volume 30mL of per 250mL shaking flask, the rotate speed of swing bed 200r/min, cultivating time of seed 18h, seed volume 13%, cultivating time 72h.The medium of shaking flask fermentation was optimized through Placket-Burman and RAS experiments. The optimal medium compositions(g/L) were determined as: barley flour 25.9, wheat bran 14.1, (NH4)2SO4 20.0, NaH2PO4 1.15, CaCO3 5.0, tween 80 1.5, FeSO4·7H2O 0.2, CaCl₂ 1.56, NaCl 1.44 and malt sugar 5.0. And theβ-glucanase activity was 307.20U/mL. On the base of shaking flasks fermentation, the enzyme production in a 5L automated jar fermentor was studied, the highestβ-glucanase activity was 323.91U/mL with 60h fermentation.It was investigated preliminarily for theβ-glucanase character by the fermentation liquid. Its optimum pH was 4.5, it could be stable during pH 3⁷; Its optimal temperature was 55℃, it could be stable under 50℃. Some ions (1mmol/L) such as Co2+, Ca2+, Fe2+ and Na+ done good forβ-glucanase activity, while the Sn2+, Pb2+ and Zn2+ were harmful for its activity. Pepsin and trypsin have no significant effect onβ-glucanase activity.The protective agents such as xanthan gum, glycerol, albumin, NaCl and CaCl₂·2H₂O could improve the stablility ofβ-glucanase. The orthogonal experiment was used to optimize the best combination of different protective agent. The result was:CaCl₂·2H₂O 2.0mmol/L, xanthan gum 4g/L and glycerol 130g/L. The complex protective agents could enhance the enzyme thermostability, and could enhance the storage stability ofβ-glucanase with 1g/L potassium sorbate, the relativelyβ-glucanase activity was 88.47% after storing at room temperature for 3 months. It was increased by 15% comparing with the control.