Sudies On Identification And Purification Of Cellulase From Aspergillus Oryzae

Six strains(NC-2,NC-5,NC-6,CXA-9,NC-10,CXA-13)of Aspergillus oryzae were preliminarily identified to produce cellulase by using substrate plate staining method.Using one of the strains,seven cellulase activity assay methods were tested.By comparing the operating difficulty and the activity,total cellulase activity was determined by FPA method.EG activity was determined by CMC-Na method,cellobiohydrolase activity was assayed by pNPC method and BG activity was determined by pNPG method.Six cell-free fermentation enzyme solutions were combined and activity of mixture was determined,the result showed that the tested mixture did not show improved activity.The activityies of six strains was compared,the strain NC-10 with high activity was selected.The fermentation medium and the inducing substances was also studied.The result showed that the optimal condition for NC-10 fermentaion was growth in DPY medium at 28 ? for 3 days and pH5.5.CMC-Na was found to be an effective inducer.For the strain NC-10,its FPA was 0.9U/mg,EG was 26.28U/mg,BG was 6.15 U/mg,CBH was 4.1 U/mg.A pure cellulase sample was obtained by ammonium sulfate precipitation and DEAE Sepharose Fast Flow ion exchange chromatography.The molecular weight of the protein was 53 kDa and showed a single peak when analysised with HPLC.However,the protein showed low cellulase activity.EG was separated from CBH and BG by using ammonium sulfate precipitation,Q Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography.In addition,the gene CBH I of Trichoderma viride and gene CBH II of Trichoderma reesei was amplified with PCR and cloned into vector successfully.