| The biomass materials such as cellulose, hemicellulose and lignin can be used as raw materials for the production of electricity, gas or liquid fuel, which is very abundant and sustainable. Specifically, to achieve the sustainable development, the prospect of nonfood-based ethanol is very great. But the cost of cellulase is too high, about50%of the nonfood-based ethanol processing costs. The crude cellulase products usually incomplete or unbalanced component, by add in β-glucosidase can reduce the cost of cellulase. In this study, we optimized the medium and fermentation conditions of the enzyme production by Aspergillus niger, shorten the cycle of enzyme production, improved the activity and decreased the production cost.To improve the beta-glucosidase enzyme activity of Aspergillus niger C112in this research, We optimized the inoculation method, and the Seed culture conditions for this method was optimized. confirmed that Culture Aspergillus niger with potato medium of60h as seed is the best condition. Optimized study was made on many single factors, including inoculation quantity, carbon and nitrogen source, initial pH, liquid volume, the fermentation temperature, rotation speed and fermentation time, etc, found that, the best condition was confirmed as follows:inoculation amount of6%, corn-core56g/L, malt extract24g/L, ammonium sulfate2.8g/L, initial pH4.87. Under this condition,60mL medium in250mL shake flask,28℃,200r/min, cultivation for120h, the β-glucosidase activity can be up to10.566U/ml,39.08%than before.The different carbon sources in different particle size affected the production of β-glucosidase by Aspergillus niger C112. We collected those datas to confirm the optimal particle size of carbon sources. Use Unsifted bran, Straw powder which particle size≤0.180mm, and corn cob which Particle size>0.180mm as carbon sources respectively. Specifically, the corn cob is the one which made highest enzyme production capacity and low cost.Box-Behnken central composite design was used to evaluate the effects of three fermentation conditions on the production of β-glucosidase by Aspergillus niger C112. Analysis the Response surface which showed that the maximum β-glucosidase yield of31.150U/ml was obtained at82.94g/L carbon source, culture at30.62℃, and initial pH of6.17, closed to the predicted maximum31.118U/ml. The prioritize order of the factors which influenced the enzyme production is as follow:the carbon source concentration> culture temperature> initial pH.During the fermentation process, we regulated the pH, which affected the production of β-glucosidase by Aspergillus niger C112. It confirmed that in Non-intervention state, the pH6.0was down to3.83Within24hours. Then it stabilized at about3.33at Last. The Aspergillus niger was high metabolism within24h to48h.But the pH regulating during the fermentation process could not improve the enzyme activity. Adjust the pH during the fermentation is unnecessarily.During the fermentation process, we regulated the culture temperature in different states, which affected the production too. It confirmed that cultured for36h at30℃early in the fermentation then drop temperature to28℃, total cultivation for144h can achieve the maximum β-glucosidase activity, about49.570U/ml.This paper also studied some properties of β-glucosidase which produced by Aspergillus niger C112. The results show that, the Km and Vmax of β-glucosidase enzymolysis the4-nitrophenyl-beta-D-glucopyranoside is1.249×10-3mol/L and0.195μmol·ml-1·min-1. Its optimum temperature is60℃and stable at this temperature.70℃and above will lead this enzyme to inactivation. |
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