| Cellulosic ethanol is a very important source of renewable biomass, which is a good alternative to solve the energy crisis. Cellulase is a key material for the development of cellulosic ethanol, however, the cost of cellulase production, low activity and instability and other issues has become the bottleneck in the development of cellulosic ethanol. Therefore, to reduce the cost of cellulase production, increase enzyme activity and enzyme stability is a key research direction of the cellulase.In this study, Arundo donax was used as materials. Penicillium decumbens was used as producing bacteria and fermentation process has been optimized for the cellulase production. First, the fermentation bacteria were purified to determine the stained bacteria as Bacillus subtilis, Pseudomonas and Staphyloccocus aureus. Tetracycline was confirmed as a good inhibition (dosage16μg/mL), and tetracycline has no effect on the growth and cellulase production of Penicillium decumbens. The purified strains was maintained a very high stability within the4th generation. The results of enzyme production curve showed that the optimal incubation time of enzyme production was84h. FPA, EG, CBH and BG were achieved to the highest value of enzyme production at84h,96h,108h and120h, with7.851IU/mL,20.814IU/mL,2.961IU/mL and3.585IU/mL activity, respectively.The results of fermentation process and conditions optimization has shown that strains in the F (2) had the highest enzyme activity of the fermentation proceeds for7.43IU/mL. When inoculum was12%, the enzyme production had highest level of7.71IU/mL. Bran was added as37.5g/L and the pore size of less than40meshes could achieve highest enzyme activity of6.79IU/mL. Ion concentration of the buffer would affect the efficiency of enzyme production, and when the concentration of sodium citrate was0.005M, filter paper enzyme (FPA) activity the highest value which19.6percent higher than the blank group activity. The highest filter paper activity achieved to8.1IU/mL when potassium dihydrogen phosphate dosage was1.5g/L. Plackett-Burman experimental results of effect on the efficiency of enzyme production in the fermentation medium showed that the factors of wheat bran, microcrystalline cellulose, initial pH were very significant factors, Tween80, KH2PO4were significant factors, and other factors were not significant. The enzyme production was increased by25%after optimization of culture medium. After the fermentation pH was controlled at5.5, the filter paper enzyme activity was20.3%higher than without pH controlled.In addition, studies the stability of cellulases produced by the fermentation has shown that the optimum temperature was60℃and the optimum pH was4.5. The stabilities of endo-glucanase (EG), exo-glucanase (CBH) and β-glucosidase (BG) were investigated and compared with that of FPA under different pH and temperature. The results indicated that CBH and FPA were more sensitive to pH and temperature than EG and BG and the stability of CBH was very similar to that of FPA under the conditions. Under the conditions of65℃the thermal stability of the BG is better than the other three types, which was incubated1h and6h maintained a87.6%and56.4%of the activity, respectively. The cellulase enzyme has a wider pH range of applications under the condition of35℃. The investigations of cellulase stabilities under different pH and temperature would be beneficial to the industrial production and application.Finally, studies on the affect stability of cellulase by Penicillium decumbens through add contrast glycol, glycerol, erythritol, xylitol, sorbitol, the result showned that sorbitol with concentration of2.5mol/L had the best effect. In addition,15%maltose cyclodextrin had the otimal effect of thermal stabilization, which has very important practical significance of the cellulase application. |
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