Identification Of A Novel Cellulase-producing, Extraction, Purification And Characteration Of Cellulases And Application Of The Fungi

A new strain which can produce cellulases with high enzymic activity was isolated from a fermented traditional food in jiangxi province. The separation and purification of the cellulases from the new strain were investigated and the enzymic properties were analyzed. The nutritional constituent alteration in the residue producing soybean food (RPSF) before and after fermentation by this new strain was studied and the extraction of the soluble glucoside was primarily conducted.The micro-morphology of mycelia and spores of the new high-yielding cellulase strain were observed. The results showed that its morphological characteristics possessed of that of Monilia sitophila Mont. This strain was named as MC-1.The studies on liquid fermentation conditions for MC-1 in 250 ml flasks were carried out through the single factor experiments. The results indicated that the culture medium components and the culture conditions had a significant influence on cellulase-producing. The optimum culture medium composition was as follows: 0.3% of mixture of straw and bagasse (the ratio of straw to bagasse was 4:1) as complex carbon source, 2% of mixture of yeast extract and peptone (the ratio of yeast extract to peptone was 1:2) as complex nitrogen source, 0.2%KH₂PO₄ and 0.08%MgSO4. The optimal culture conditions for producing cellulase were 10ml of the spore inoculum, initial pH 4.8, constant temperature 28³0°C and cultivation time 6 days.The separation and purification of cellulases was conducted by saturated-precipitation method using 20-65% ammonium sulfate, Sephadex G-200 and DEAE-Sephadex A-25 Chromatography. Three components, cellulases Cl, Cx and p-G were obtained and their molecular weights were 31.9 KD, 64.8 KD and 22.1KD respectively by SDS-PAGE in comparison with the standard protein. The analysis of enzymic properties demonstrated that the optimum temperature was 55°C, 45°C and 60°Cfor Cl, Cx and β-G respectively and the optimum pH value was 4.5, 5.0 and 4.5 respectively. Metallic ions had some influence on the enzymic activity. The enzymic activities of the three cellulases were remarkably stimulated by the addition of Mn²⁺ at 1¹0mmol/L, activity of cellulose Cl was increased by 150.8-955.3%, Cx by 117.4³00.8%, and P-G by 106.1—737.8%. The Cl and Cx activities were inhibited by Li⁺, while p-G activity was stimulated slightly. Cu²⁺, Al³⁺, Zn²⁺, Ca²⁺and Cr³⁺ inhibited all three cellulases activities.The changes of isoflavone, crude fibers, proteins and water-soluble solids in RPSF by solid fermentation with MC-1 were investigated. The results showed that the content of crude fibers in the fermented RPSF decreased by 35.2% (23.8% vs 35.5%), while the content of the water-soluble solids, isoflavone and proteins was increased by 5 times (36.98% vs 7.54%), 10% (0.66 vs 0.60 mg/g) and 32% (32.59% vs 24.66%) respectively, compared to the unfermented one. It testified that the nutritional and functional components of RPSF could be considerably altered by MC-1 fermentation and the processing behavior would be improved.By using single factor gradient experiments, the optimal conditions for...