|其他摘要||Filamentous fungus Trichoderma reesei (Tr. reesei) is a very efficient cellulase expression system, and also the most widely studied cellulase system at present. Tr. Cel7B (also known as EGI) is a major endoglucanase secreted by Trichoderma reesei. The amount of expressed Tr. Cel7B accounts for 5-10% of the total cellulases. The molecular weight of Tr. Cel7B is about 50 KDa, with the isoelectric point of 4.5. Usually, the enzymatic hydrolysis of cellulose is divided into the following steps: Firstly, the enzyme adsorbs to the surface of cellulose; Secondly, the enzyme moves along the surface of cellulose to identify the chain end; Thirdly, the glycosidic bond of cellulose is cut and the product released (mainly cellobiose); Finally, the cellulase either slides along the surface of cellulose, or desorbs from cellulose. However, the enzymatic hydrolysis of cellulose is inefficient currently, and is not well adapted to the conditions of industrial production whereas the recovery efficiency of the enzymes is very low and costly. From the microscopic point of view, the cellulose hydrolysis mechanism by a single enzyme at the molecular level is not very clear. Therefore, it is very important to conduct a thorough study on the cellulases catalyzed hydrolysis, and to improve the activity of cellulases and their environmental adaptability.
A structural model of Trichoderma reeseiCel7B (Tr. Cel7B-CD) catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. However, a further study shows that this slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amounts of reducing sugars released after 24 h hydrolysis of phosphoric acid swollen cellulose, Avicel, and filter paper, are correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ~15-35% more reducing sugars than the wild type Tr. Cel7B-CD in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose.
In addition, we also studied the thermalstability of Tr. Cel7B-CD. For proteins that denature irreversibly, the denaturation is typically triggered by a partial unfolding, followed by a permanent change (e.g., aggregation). The regions that initiate the partial unfolding are named “weak spots”. In the thesis, a molecular dynamics (MD) simulation and data analysis protocol is developed to identify the weak spots of Tr. Cel7B-CD, through assigning the local melting temperature (Tmp) to individual residue pairs. To test the predicted weak spots, a total of eight disulfide bonds were designed in these regions and all enhanced the enzyme thermostability. The increased stability, quantified by ΔT50 (which is the T50 difference between the mutant and the wild type enzyme), is negatively correlated with the MD-predicted Tmp, demonstrating the effectiveness of the protocol and highlighting the importance of the weak spots. Strengthening interactions in the weak spots proves to be a useful strategy in improving the thermostability of Tr. Cel7B-CD. These studies will help understand the catalytic mechanism of cellulase hydrolysis at the molecular level, provide useful information for improving its activity by the subsequent protein engineering which is important for the industrial application.|