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Tr. Cel7B-CD对纤维素的水解机理及其热稳定性研究
张树军
学位类型博士 ; 学位论文 ; 博士 ; 学位论文
导师姚礼山
2016-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物化学与分子生物化学
关键词内切葡聚糖酶 水解 吸附 热稳定性 分子动力学模拟
摘要丝状真菌里氏木霉(Trichoderma reesei,简写为Tr. reesei)是一个非常高效的纤维素酶表达系统,也是目前研究最为广泛的纤维素酶体系。Tr. Cel7B(又称EGⅠ)是里氏木霉分泌的主要内切葡聚糖酶,也是降解纤维素最重要的内切酶之一,其表达量占总纤维素酶表达量的5-10%。Tr. Cel7B分子量约为50kDa,等电点是4.5。通常将酶水解纤维素底物的过程分为以下几个步骤:首先,酶吸附到结晶纤维素表面;其次,酶在纤维素表面移动以识别纤维素链末端;然后,水解糖苷键释放产物(主要是纤维二糖);最后,产物从终端释放,纤维素酶在纤维素表面向前滑移然后剪切下一个糖苷键或者从纤维素上解吸附。然而,从宏观上看,目前降解纤维素的酶系统水解效率不高,还不能很好的适应工业生产的条件,并且酶回收效率低,成本较高,成为限制纤维素酶在工业领域广泛应用的一大瓶颈;从微观上看,单个酶在分子水平上水解纤维素的作用机制不是很清楚。因此,对纤维素酶的水解机理进行深入研究,结合相关技术对其进行合理设计和改造,以提高纤维素酶的活性和环境适应性显得尤为重要。 我们用分子动力学模拟的方法,构建了里氏木霉内切葡聚糖酶Cel7B-CD(Tr. Cel7B-CD)的催化域结合到纤维素表面的结构模型,并预测和实验验证了Tr. Cel7B-CD上结合到纤维素表面相关的重要残基。研究发现,构建的Tr. Cel7B-CD 的13个突变体对磷酸溶胀结晶纤维素(PASC)和滤纸(FP)的吸附特征明显不同。尽管这些突变体对滤纸的吸附能力比磷酸溶胀结晶纤维素的要弱10倍左右,但是突变体对两种底物的吸附能力成正相关,通过酶动力学研究发现,Tr. Cel7B-CD催化两种底物的水解反应都存在反应速度迅速下降的现象,然而,进一步研究发现,速度的下降和酶与纤维素结合常数没有明显关系,而与酶的起初速度成负相关。同时,本文也研究了Tr. Cel7B-CD催化磷酸溶胀结晶纤维素、结晶纤维素(Avicel)、滤纸和可溶底物p-硝基苯基乳糖苷(PNPL)时的还原糖释放量,发现反应24小时后 Tr. Cel7B-CD水解3种不溶底物(磷酸溶胀结晶纤维素、结晶纤维素和滤纸)释放的还原糖量与水解可溶底物释放的还原糖量成正相关。13个突变体中,与野生型Tr. Cel7B-CD相比,6个突变体(N47A、N52D、S99A、N323D、S324A和S346A)对磷酸溶胀结晶纤维素和滤纸的还原糖释放量提高了15-35%。上述研究表明,里氏木霉来源的内切葡聚糖酶Tr. Cel7B-CD在催化水解纤维素时,反应速度的下降并不决定于酶吸附纤维素的能力。 此外,我们还研究了内切葡聚糖酶的热稳定性。在引起蛋白不可逆失活的原因中,一个典型的原因是蛋白结构的部分解折叠,进而引起蛋白结构永久改变。通常这些最初开始解折叠的区域被称为“弱点”区域。Tr. Cel7B-CD是一个水解纤维素的重要内切葡聚糖酶,在本研究中,利用分子动力学模拟和数据分析工具,我们找到了里氏木霉来源的内切葡聚糖酶Tr. Cel7B-CD的“弱点”区域,预测了“弱点”区域对应残基对的区域熔融温度Tmp,并在这些区域共引入了8个二硫键来增强酶的热稳定性。通过实验测定Tr. Cel7B-CD突变体和野生型之间的T50变化(ΔT50),发现8个突变体的热稳定性都有不同程度的提高,通过突变体之间的叠加组合,最终将Tr. Cel7B-CD的热稳定性提高了10.9℃;并且这8个突变体的ΔT50与对应的Tmp成负相关,这表明预测结果和实验结果一致。证明利用分子动力学模拟技术预测“弱点”区域,进而“加固”这些区域,从而提高酶的热稳定性这一方法是非常有效可行的。而这些研究将有助于在分子层面上理解纤维素酶的催化水解机制,为后续的蛋白质工程研究提高其活性提供有用信息,并将对纤维素酶工业化应用有重要意义。
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.
作者部门仿真与模拟团队
学科领域分子生物学
公开日期2016-06-30
学位类型博士 ; 学位论文 ; 博士 ; 学位论文
语种中文
文献类型学位论文
条目标识符http://ir.qibebt.ac.cn/handle/337004/9768
专题仿真与模拟团队
作者单位中国科学院青岛生物能源与过程研究所
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张树军. Tr. Cel7B-CD对纤维素的水解机理及其热稳定性研究[D]. 北京. 中国科学院大学,2016.
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