Study On Breeding Of β-mananase Overproducing Strain, Purification And Properties Of β-mananase

By purification and rejuvenation to available CXJZ series strain, and comparison of clearing halos in selecting culture plate andβ-mannanase activity in fermented liquid, CXJZ11-01 was acquired, which secretsβ-mannanase most of all in studied strains.Conjunctive with mono-factor and multi-factor statistical experiments, the ingradients of optimum growth medium and optimal fermentation medium for CXJZ11-01 were obtained. Growth medium is 0.5% NaCl, 0.1% glucose, 0.4% konjac powder, 0.3% beef extract, 0.2% yeast extract paste, and 0.5% peptone. Fermentation medium is 0.5% NaCl, 0.7% konjac powder, 0.3% beef extract, 0.2% yeast extract paste, and 0.5% peptone.By systematically studying the way ofβ-mannanase detection and enzyme production law, it is concluded thatβ-mannanase activity from CXJZ11-01 reaches to 3050U/ml, when it has been cultured for 9h in optimal condition. Besides, the measurement system ofβ-mannanase activity is ascertained that the optimal pH is 6.0, optimal temperature is 65℃, optimal substrate is 2mg/ml konjac glucomannan gum, and that the buffer is 0.05mol/L Citric Acid-0.1mol/L Disodium Hydrogen Phosphate.By ultra-filtration and gel column chromatography,β-mannanase secreted by CXJZ11-01 was purified. The results are as follows.(1) The suitable membrane dimension is 10～50kD, condensity ofβ-mannanase activity is 30.87, recovery rate is 63.42%, and the purification multiple of protein is 10.(2) 13 fractions are separated from concentrated solution of ultrafiltration by Sephadex G-100 column chromatography, but only one showsβ-mannanase activity.(3) Single dominant band is detected in SDS-PAGE.Therefore, singleβ-mannanase can be obtained by a simple flow-sheet, membrane separation technique adding to chromatography.By studying the purifiedβ-mannanase from CXJZ11-01, its physicochemical properties are as follows.(1) The molecular weight ofβ-mannanase is 28.2 kD.(2) The stable pH ofβ-mannanase is 4.5～7.0, and the thermostable temperature is below 70℃.(3)β-mannanase is activated by 1mmol/L Mn²⁺, Cu²⁺, Zn²⁺, Ca²⁺, Mg²⁺ and Al³⁺, while inhibited by the same concentration of Ba²⁺ and Pb²⁺.(4) When the substrate ofβ-mannanase is locust bean gum, the Km and the Vm are 7.407mg/ml and 2000μmol/(min*ml), respectively.(5) When the substrate ofβ-mannanase is konjac glucomannan gum, the Km and the Vm are 1.706mg/ml and 588.2μmol/(min*ml), respectively.