| 其他摘要 | 里氏木霉纤维素内切酶EGI通过与其他纤维素酶（外切酶,β-葡萄糖苷酶等）协同作用一起催化水解结晶纤维素。在里氏木霉纤维素酶体系中，EGI占5%-10%，是重要的纤维素内切酶。本文以里氏木霉纤维素内切酶EGI的催化域(CD)为研究对象，采用分子生物学方法，进行了该基因的克隆并在大肠杆菌Origami(DE3)里面表达，重组蛋白的纯化及其酶学性质研究，通过定点突变的方法，揭示了催化中心邻近的几个重要氨基酸残基对其催化效率的影响。同时，本文还通过引入二硫键的方法，对其热稳定性的提高进行了探索，找到了几个二硫键突变体及组和突变体。具体结论如下：1. 分析里氏木霉EGI的晶体结构，发现Y146, Y170, D172和R108这几个氨基酸残基距离EGI催化中心很近，它们很可能与催化中心产生氢键相互作用，形成氢键网络，对催化效率产生重要影响。本文通过对这几个重要氨基酸残基进行保守突变并进行酶促动力学实验，发现四个突变体的催化效率相比野生型有不同程度的降低。其中R108K降低的最少，表明它对催化效率的影响最小；Y146F和D172N降低的程度相当；Y170F则降低的最多，说明这一位点对催化效率的影响最大。对突变体及野生型蛋白进行分子动力学模拟计算结果表明，突变体催化中心E197残基上的Oε2与底物pNPL上的C1B的距离D1，以及催化中心E202残基上的Oε2与底物pNPL上的O4C的距离D2相比野生型均有不同程度的变化。这两个距离在一定程度上体现了酶催化纤维素水解过程中质子转移和亲核进攻的难易程度，从而影响反应能垒。R108K的D1与野生型相差不大，另外三个突变体的D1就比野生型的高很多，其中高出最多的是Y170F，其次是Y146F和D172N。而对于D2，除了D172N比野生型要低一些以外，其他几个突变体的D2均比野生型的高，其中高出最多的是Y170F，其次是R108K和Y146F。从计算结果可以看出，总体而言，突变体的D1, D2比野生型的大，表明他们参与反应时，形成过渡态时比野生型要难，因此其催化效率应该比野生型低，这解释了实验观察到的现象。2. 本研究还通过实验验证，找到了几对可能形成二硫键的位点进行突变，并比较了突变体与野生型的热稳定性。结果表明有几对二硫键突变体对羧甲基纤维素的热稳定性均有所提高，其最适温度分别从野生型的50℃左右提高到了55℃, 55℃, 60℃, 70℃左右。; Endo-β-1,4-glucanases (EC 3.2.1.4)and other cellulases from the cellulolytic fungus Trichoderma reesei, including exo-β-1,4-glucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21), play a synergistical role in the degradation of crystalline cellulose. Endoglucanase I (EGI) is the major endoglucanase produced by Trichoderma reesei, accounting for 5 to 10% of the total amount of cellulases produced by this organism.In this thesis, we cloned the gene of EGI catalytic domain and expressed in Escherichia coli Origami(DE3). Its catalytic property is also investigated. By using site-directed mutagenesis, we revealed the impact of four important amino acids close to the catalytic core on the catalytic efficiency. Meanwhile, we explored the improvement of its thermostability by adding new disulfide bridges and found four favorable ones. The results are showed as follows:1. By analyzing the crystalline structure of EGI from Trichoderma reesei, we found that four amino acids Y146, Y170, D172 and R108 located very near to the catalytic core and might have an important effect on the catalytic efficiency by forming a hydrogen-bond network. Conservative mutations on these four residues and kinetic experiments were performed. The results showed all the four mutant proteins had a lower catalytic efficiency compared with the wild type. Among them, R108K had a least degree of efficiency loss. Y146F and D172N had a similar reduction in catalytic efficiency, illustrating that they had a comparable effect on the catalytic efficiency. Y170F had the most degree of reduction and implicated it had a considerable effect on the catalytic efficiency. Molecular dynamics simulation is also used in this research. The result showed that the distance between Oε2 of catalytic core residue E197 and C1B of pNPL, namely D1, and that between Oε2 of catalytic core residue E202 and O4C of pNPL, namely D2, varied greatly between wild type and mutant proteins. The D1 of R108 is similar to that is wild type, while other three mutant proteins had a much bigger D1 than wild type. The most significant is Y170F, followed by Y146F and D172N. As for D2, the mutations’ are bigger than wild type, except for the D172N. The biggest is Y170F, with R108K and Y146F followed behind. Overall, the result demonstrated that the mutant proteins had bigger D1 and D2 than wild type, and the formation of transition state is harder for them in reaction and thus they had a much lower catalytic efficiency than wild type. The result agreed well with the experiment data.2. In this research, through verification by experiment, we found several pairs of sites that might form disulfide bridges and site-direct mutated them. By comparing their thermostability, we found four mutations were more thermostable than wild type. Their optimum reaction temperatures on CMC were about 55℃, 55℃, 60℃ and 70℃ separately, while that of the wild type was only about 50℃. |
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