Study On The Production Of Thermostable β-glucanase By Biological Methods Using Beer's Spent Grains

β-glucanase is an important industrial enzyme that may largely eliminate the negative effects caused by cerealβ-glucan on the processing of beer and feed industries. So it plays an especially important role in beer and feed stuff industries. But, the study on theβ-glucanase is still in the beginning and the thermostability ofβ-glucanase was insufficient when compared with foreign studies. The poor thermostability ofβ-glucanase does not satisfy the temperature requirement of malting and feedstuff preparation, so it is significant to improve the thermostability ofβ-glucanase.In this study, an isolate of X-5 from soil was found producing a thermostableβ-glucanase. After mutated by mutagens such as UV and DES on the strain, a high yield producing thermostableβ-glucanase strain Bacillus subtilis AS35 was obtained. Production of this enzyme was investigated by optimizing components of medium and conditions of fermentation using single factor experiments and Response Surface Methodology(RSM). Theβ-glucanase of AS35 was purified primarily and characterized for its properties and kinetics. At last, the effects of adding the complex protective agents on the thermostability and storage stability ofβ-glucanase were studied. The major results were summarized as follows:1. A thermostableβ-glucanase producing strain X-5 with aβ-glucanase activity level of 8.64 U/ml at 37℃for 60h was isolated from numerous soil and identified as Bacillus subtilis.2. The isolate was then mutated by exposure to ultraviolet, followed by chemical pressure of diethyl sulfate. The productivity ofβ-glucanase of the selected mutant Bacillus subtilis AS35 was improved greatly by mutated, which was 15.83 U/ml at the same conditions, 1.83 times higher than that of the original strain X-5. Subculture test indicated that the hereditary character of mutant AS35 was stable.3. In this paper, the medium composition and the fermentation conditions were investigated. An optimized medium for the production of the enzyme by the mutant was composed of beer lees flour 47.476 g/L, corn flour 15.0 g/L, peptone 11.539 g/L, (NH₄)₂SO₄ 3.0 g/L, K₂HPO₄ 2.310 g/L, CaCl₂ 1.0 g/L, NaCl 5.0 g/L, MgSO₄·7H₂O 0.4 g/L, FeSO₄·7H₂O 0.01 g/L and Tween-80 0.096 ml/100ml, which was obtained through single factor experiments and Response Surface Methodology(RSM) design experiments. Verification experiment results showed that theβ-glucanase activity (30.66U/ml) was similar to that (31.33U/ml) predicted by model, and the relative error was 2.13% between them. The optimal conditions for fermentation were determined as temperature 36℃, initial pH 7.0, 12.0 % (v/v) seed culture, inoculum age 18 h, charge quantity 50ml medium in 250ml shaking flask and rotating rate 210 r/min. A 60h incubation of the mutant in the liquid medium under the optimized conditions resulted in aβ-glucanase activity level of 32.12 U/ml, which was 1.0 times more than that in the original flasks.4. It was investigated preliminarily for theβ-glucanase character by the fermentation liquid. The purifiedβ-glucanase had maximal activity at pH 5.5 and its relative activity exceeded 85% after maintenance at pH 4.0-7.0 for 24 h at 4℃. Its optimal reaction temperature ranged from 55 to 65℃with maximal activity at 60℃. Its activity could be suppressed at the presence of Cu²⁺ and Zn²⁺, especially by Pb²⁺, Fe³⁺ and A1³⁺; but conspicuously stimulated by Fe²⁺, Co²⁺ or Ca²⁺, especially by Co²⁺. However, other metal ions including K⁺, Na⁺, Mn²⁺ or Mg²⁺ had little effects on its activity.5. The protective agents such as xanthan gum, glycerol and CaC1₂ could improve the stability ofβ-glucanase. The orthogonal experiment was used to optimize the best combination of different protective agent. The result was: CaC1₂ 2.0mmo1/L, xanthan gum 3 g/L and glycerol 30g/L. The complex protective agents could enhance the enzyme thermostability, and could enhance the storage stability ofβ-glucanase with l g/L sodium benzoate, the relativelyβ-glucanase activity was 85.23% after storing at room temperature for 1.5 months. It was increased by 15% compared with the control.