| Cellulase is widely used in food industry, wine-making, textiles industey, agriculture and other area. It is responsible for conversion of renewable cellulosic biomass to simple sugars for fermentation to ethanol by anaerobic bacteria, which can settle the issue of renewable energy and environmental pollution etc. But its application is restricted because of its lower expression and specific activity. Cel5G is a novel endo-β-1,4-glucanase discovered by our laboratory. It is classified into Glycoside hydrolase family5. In this thesis two aspects of cel5G gene have been studied, one is gene modification and the other is expression in Pichia.Cellulose binding domain(CBD) can modify the nature of the cellulose, And promote the association of the enzyme with the substrate and play the central role in the enzymatic hydrolysis of plant cell wall. cel5G gene and two CBD genes have been fused,one is from Trichoderma reesei CBH I and other one is from Clostridium cellulovorans cellulose combined protein(CbpA). Both of the recombination genes were expressed in E.coli BL21successfully. The combination and hydrolysis of microcrystalline cellulose were studied by using the crude enzyme. Therecombination enzyme consisted of Cel5G and CBDH1can combine the microcrystalline cellulose effectively. And3-fold higher to hydrolysis the microcrystalline cellulose compared with the original enzyme. On the other side the recombination enzyme consisted of Cel5G and CbpA don’t have these characteristics.The enzyme expressed by E.coli is easy to form inclusion body and hard to be purified.In contrast of E.coli expression system, Pichia pastoris has many merits in the gene expression, protein process, secretion, post-translation modification and the glycosylation modification, which means it is more suitable for industrial production. The gene cel5G has been cloned into vector pEFaA, constructed a yeast integrative expression plasmid,and expressed in Pichia pastoris GS115.In according to hydrolysis reaction and SDS-PAGE, the cel5G gene was expressed successfully in Pichia pastoris and its easy to be purified. Cel5G exhibited highest activities at pH4.8and50℃in50mM citrate buffer, it is stable over a wide pH range from3.0to10.0and can still maintain80%of the enzymatic activity in50℃for30mins. |
