| Endo-β-l, 3-1, 4-glucanase is an important industrial enzyme that eliminate the negative effects caused by cereal β-glucan in brewing and feed industry. In this study, thermostable β-glucanase producing strain was screened and its characteristics for enzyme production was studied. Medium and conditions of fermentation was optimized using respond surface methodology(RSM). β-glucanase was purified and properties and kinetics was studied. The batch process and dynamics model was studied and medium-scale up was investigated. The partitioning behavior of β-glucanase in different aqueous two-phase systems was investigated. The application effects of enzyme preparation in mashing process and kinetics for degradation of β-glucan in wort was studied. The main results were as following:1. The p-glucanase producing strain was isolated from soil and the optimal temperature of p-glucanase was 65℃ and it was identified as Bacillus subtilis. The productivity of β-glucanase of B. subtilis mutant ZJF-1A5 was improved greatly after mutated by ultraviolet and diethyl sulfate, 2.42 times higher than that of initial strain. Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of B subtilis ZJF-1A5 and p-glucanase production. Yeast extract was the best nitrogen source, the following was soybean flour. All of inorganic nitrogen chosen in study were not good for cell growth and enzyme production. KH2P04, CaCl2 concentration influenced the cell growth and enzyme production, while MgS04-7H20 concentration had little effects. Charge quantity affected p-glucanase production significantly also. Adding barley p-glucan in medium was benefit to β-glucanase production. The composition of fermentation medium optimized with RSM was(g/L): dextrin, 38.00; yeast extract, 29.91; barley flour, 12.00; KH2P04, 1.34; MgS04-7H20, 0.1; CaCl2, 0.432. β-glucanase activity of broth was 235. 87 U/mL at 48 h using optimized medium, similar to that predicted by model. The β-glucanase, α-amylaseand neutral protease produced by B subtilis ZJF-1A5 were associated partially with cell growth, enzymes activities increased following the cell growth and increased significantly when cells entered stationary phase.2. β -glucanase of B. subtilis ZJF-1A5 was purified by Sephadex G-100 and DRAR-Collulosc 52 chromatography, and its properties and kinetics was studied. Its optimum pH value is 6. 5, and its optimum temperature is 65癈. The thermostability of enzyme was influenced by Ca2+ and substrate, Ca2+ and substrate can increase half life of P -glucanase. Metal cation influence enzyme activity, Fe3+and A13+ inhibited enzyme activity, K+, Na+, Mn2+, Mg2+ have insignificance effects on enzyme activity and Fe2+, Co2+, Ca2+, especially Co2+, have active effects on enzyme activity. K. and V.M of P -glucanase for barley P -glucan as substrate was 3.855 mg/mL and 0.1248 mg/ (mL ?min )respectively, kinetic model was and Km and Vmax forlichenan as substrate was 6.351 mg/mL and 0.1951 mg/ (mL*min)respectively, kinetic model was 3. The agro-industrial by-product was used to optimum fermentation in order to decrease the cost. The concentration of barley flour, corn flour and soybean flour influenced the P -glucanase production signi ficantly. Medium optimized wi th RSM was(g/L) : barley flour, 63. 5; cornflour, 44.8; KH2P04, 1.0; MgS04-7H20, 0. 1 ;CaCl2, 0. 1. p-glucanase activity of broth was 250. 73 U/mL at 48 h using optimized medium.The composition of inoculum medium optimized with RSM was(g/L): dextrin, 35.10; yeast extract, 34.80; KH2P04, 4.60; MgS04-7H20, 0.1; CaCl2, 0. 35. The maximum biomass predicted by model was 8. 36 g/L. The biomass was 8.21 g/L cultured at 20 h. Inoculum age and size influenced the p-g]ucanase production and fermentation process.The optimal temperature for p-glucanase production was 35-37℃ and optimum charge quantity was 30 mL medium in 250 mL flask. Adding adequate oxyg...
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