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β-glucanase is an important industrial enzymes, which can effectively eliminate the negative impact of the cereal β-glucan in beer and feed production. Therefore, β-glucanase in beer and feed industry plays an important role. Compared with foreign countries, the thermal stability of the late start, our study of β-glucanase and β-glucanase developed a generally poor and can not meet modern beer saccharification process and pellet feed granulation temperature requirements seek high-yielding strains of thermophilic β-glucanase has been the focus of attention and research direction. Accordingly, the increase of β-glucanase of thermal stability has a very important significance. The papers from the bacteria Isolation and Screening start producing thermophilic β-glucanase activity through physical, chemical mutagenesis breeding mutant AS35 and study the characteristics of its enzyme production by single factor experiment and response surface method to optimize the shake flask fermentation conditions for enzyme production, preliminary separation and purification of β-glucanase, reveal the enzymatic properties of the crude enzyme solution, last discussed adding a protective agent crude enzyme solution thermal stability and storage stability impact, provide a theoretical basis and reference for industrial production and application. The main conclusions are as follows: 1, isolated from the Turpan heat collected soil samples screened a production a higher thermophilic β-glucanase activity of the wild-type strain X-5, 37 ° C fermentation 60 h fermentation fluid activity of up to 8.64 U / ml. As Bacillus subtilis (Bacillus subtilis), initially identified by morphological and biochemical identification of the wild-type strain X-5. Fermentation activity of mutant AS35, ultraviolet radiation and dimethyl sulfate has ester compound mutagenesis of the wild-type strain X-5 enzyme production levels were significantly increased under the same conditions of 15.83 U / ml, starting strain X-5 1.83 times the passage test to prove the mutant strains producing enzyme performance is relatively stable. 3, through the single factor experiment and response surface experiment, their shake flask fermentation medium optimized production thermophilic β-glucanase best medium composition (g / L): bad wheat flour 47.476, corn powder 15.0, peptone 1,153.9 (NH 4 ) 2 SO 4 3.0 K 2 the HPO 4 2.310, CaCl 2 1.0, NaCl 5.0, MgSO 4 · 7H 2 O 0.4, FeSO 4 · 7H 2 O 0.01, Tween-80 0.096 ml/100ml. The validation experiments show that β-glucanase activity reached 30.66 U / ml, with the predicted results of 31.33 U / ml close relative error of 2.13%. On this basis, optimized by single factor experiment to study the fermentation of the impact of environmental factors on the strains AS35 enzyme production capacity. Optimization results for: seed age 18h, inoculum size 12% 50 m1/250 m1 to shake flask shaker at 210 r / min, incubation temperature 36 ° C, initial pH 7.0, the optimum fermentation time 60h. The highest activity of β-glucan in the optimal conditions, the fermentation broth reached 32.12 U / ml (15.83 U / ml) than before the process optimization to improve nearly doubled. 4: enzyme between 55 ℃ -65 ℃ higher enzyme activity optimum reaction temperature of 60 ° C; optimum pH of 5.5, pH4.0-7.0 range than the nature of the enzyme high stability, stored at 4 ℃ were more than 85% of the residual activity after 24h; lmmol / L concentration, Fe 2 , Co 2 , Ca, 2 , the activation especially Co 2 on the enzyme activity; K , Na , Mn 2 , Mg 2 has little effect on the enzyme activity; Cu 2 , Zn 2 slight inhibition of enzyme activity role; Pb 2 , Fe 3 , A1 3 significantly inhibited the activity of enzyme activity is almost completely suppressed. 5, xanthan gum, glycerol, and CAC1 2 -protecting agent such as, to some extent, improve the thermal stability of the enzyme solution. By orthogonal experiment and validation experiments, the final choice of the composition of xanthan gum 3 g / L, of CaCl 2 2.0 mmol / L and glycerol 30 g / L composite protective agent. The composite protective agent to improve the thermal stability of the enzyme, the enzyme viability loss range from 50 ℃ -70 ℃ increased to 55 ° C to -70 ° C; composite protective agent and 1 g / L sodium benzoate is used in conjunction with the enzyme solution can significantly improve the storage stability, and placed at room temperature for 1.5 months, the relative residual activity of up to 85.23%, and increased by about 15.0% than the control group.
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