| In this thesis, microcrystalline cellulose and starch hydrolyzate were chosen as carbon source for cellulase production by Trichoderma reesei Rut C30. During fed-batch cultivation of the microorganism, cellulase production production with enzyme recovery was performed in order to enhance cellulase production. The main research results are as follows:Starch hydrolyzate contains large amounts of fiber oligosaccharides such as cellobiose, gentibiose. These oligosaccharides have good effects on cellulase production by Trichoderma reesei. When fed-batch culitivation was conducted, starting carbon was starch hydrolyzate 6.5 g/L and the optimum daily feeding rate was cellulose 4 g/L; starch hydrolyzate 1 g/L; ammonium sulfate 1.83 g/L and urea 0.3 g/L. Under this condition, the produced cellulase had a filiter paper activity of 12.75 FPU/ml and ?- glucosidase activity of 2.54 U/mlThe enzyme recovery was helpful to promote the cellulase production. After three days of fed-batch cultivation, cellulase was partially recovered at intervals of 1, 2 or 3 days. Significant increases in cellulase(25–35%) and ?-glucosidase(over 60%) production were realized. Making up the system with different filled liquid(water, 0.05 M citrate buffer and mineral solution) after the enzyme recovery. when the enzyme recovery was 15% per day at various intervals, the total gained cellulase was 25–35% higher than fed-batch. the total gained ?-glucosidase activity increased significantly when buffer or mineral solution was used, but decreased when water was used. It may be that ?-glucosidase is sensitive to pH levels and nutrients. The citrate buffer and mineral solution could control pH effectively and supply the appropriate nutrients to enhance ?-glucosidase synthesis.Mycelia could also be removed to avoid infection if cell concentrations were maintained in a reasonable range. When enzymes were recovered every day with 15% of the enzyme, the cellulase production increased by 35.4%(without mycelia removal) and 32.5%(with 15% mycelial removal) compared with the normal fed-batch cultivation. The total ?-glucosidase activity increased over 60%. 3–4 g/L of the concentrations of cells is reasonable level in the present study. The beauty of this concept is that it not only enhances enzyme productivity but also reduces redundant fungal cells that consume carbon and nitrogen sources for survival.To evaluate the hydrolytic ability of various cellulase preparations, dilute HCl-pretreated corn stover was enzymatically hydrolyzed for 48 h by three enzyme preparations produced in the present study and a commercial cellulase. Compared with the 62.0% yield of the commercial enzyme The cellulase produced from fed-batch cultivation without enzyme recovery obtained a hydrolysis yield of 86.4% and enzyme recovery coupled with mycelia removal resulted in hydrolysis yields of 82.7% and 82.8% in 48 h. A high hydrolytic ability of the produced cellulase was also detected. Enzymatic hydrolysis was conducted at the solid-liquid ratio of 10%, together with cellulase 20 FPU/g cellulose,?- glucosidase 20 FPU/g cellulose. The enzyme hydrolysis yield was 80.47%. PEG20000 also had good effects on enzymatic hydrolysis. |
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