QQ客服 官方微博 反馈留言

题名	里氏木霉内切纤维素酶Cel5A催化机理研究
作者	舒志愚
学位类别	博士
答辩日期	2015-05
授予单位	中国科学院研究生院
授予地点	北京
导师	姚礼山
关键词	内切葡聚糖酶 吸附 动力学参数 滑移 协同
学位专业	生物化学与分子生物学
中文摘要	里氏木霉（Trichoderma reesei, Tr）纤维素酶由外切葡聚糖酶，内切葡聚糖酶，β-葡萄糖苷酶以及一些起辅助作用的氧化还原酶组成。外切葡聚糖酶从纤维素分子的两端以二糖为单位剪切纤维素，内切葡聚糖酶则随机的在纤维素链上进行剪切，主要作用在纤维素的无定形区域，β-葡萄糖苷酶则将二糖水解为单糖分子，和氧化还原酶随机的剪切糖链。本文对里氏木霉来源的内切葡聚糖酶Tr. Cel5A的催化区（Catalytic Domain，CD）水解纤维素机理进行了研究，具体包括酶与纤维素的吸附、反应动力学、滑移、以及Tr. Cel5A CD与外切酶Tr. Cel7A的协同效应等。 首先，结合计算模拟，设计突变体改变酶与底物磷酸溶胀纤维素（PASC）的吸附能力并探讨吸附与催化的关系。结果表明Tr. Cel5A CD野生型及19个突变体的吸附常数Kr值在0.12-0.39 L/g间波动，野生型的Kr值为0.26 L/g。其中，T18，K26，Y28，H229和T300这几个残基对于Tr. Cel5A CD与底物PASC的吸附起很重要的作用；Tr. Cel5A CD与PASC的吸附存在高活性-低容量的吸附，即底物中存在一些高吸附能力的位点，更容易与Tr. Cel5A CD发生作用。突变体对PASC水解活性也有变化，虽然这些突变体都集中在Tr. Cel5A CD与纤维素作用的吸附面上，但是突变以后，酶的剪切能力也发生了变化，可能是突变扰动了活性中心的结构导致的。实验测得Tr. Cel5A CD与PASC的吸附强弱和酶对PASC的活性间没有明显的相关性。 Tr. Cel5A CD与PASC反应时，随着反应的进行，反应速率在衰减，并且各突变体及野生型间的衰减速度不同。我们对Tr. Cel5A CD与PASC的反应动力学参数进行了测量，结果表明反应初始速率A与表示速率衰减的指数因子b间呈负相关，表明反应越快，反应速率下降得也越快，导致这种衰减的原因可能是酶被底物上的障碍物困住，或者是底物的不均一使底物的反应性有差别，即底物上可能存在容易被反应的部分。对吸附常数Kr与b进行拟合发现，二者间无明显相关性。表明酶反应速率的衰减不是由于吸附差异所导致。Tr. Cel5A CD对PASC的反应初始速率与Tr. Cel5A CD与对硝基苯酚纤维素二糖（PNPC）的比活间存在正相关，即Tr. Cel5A CD对可溶底物PNPC的剪切能力越强，其与不可溶底物PASC反应时初始反应速率越大。 我们还通过引入突变体改变了Tr. Cel5A CD的滑移能力。结果表明野生型及针对滑移设计的突变体在底物PASC上的滑移能力有一定差异，其中Y28A和W185A的滑移能力比野生型有提高，其余都降低，S187A的滑移能力最弱。对滑移能力进行突变以后，不仅改变了滑移能力，同时各突变体的活性也都有了不同程度的降低。Tr. Cel5A CD与PASC反应中加入Tr. Cel7A后，水解效率大幅提升，Tr. Cel5A CD与Tr. Cel7A在水解PASC时存在正协同效应，二者比例为3：7时协同最强。对Tr. Cel5A CD的滑移与Tr. Cel5A CD和Tr. Cel7A的协同度进行拟合发现，二者呈负相关，表明滑移能力越强的突变体，其与Tr. Cel7A的协同度越低。同时，加入Tr. Cel7A后，协同效应使Tr. Cel5A CD的滑移突变体及野生型间的活性差异减小。
英文摘要	The cellulases consist of exoglucanases, endoglucanases, β-glucosidases and additional oxidoreductases. Exoglucanases cleave the cellulose strand from the reducing or non-reducing end, endoglucanases cleave the strand in the amorphous region of cellulose, β-glucosidases convert cellobiose into glucose monomers and oxidoreductases cleave cellulose strand randomly. In this thesis, we studied the adsorption of Trichoderma reesei. Cel5A catalytic domain (Tr. Cel5A CD) to phosphoric acid swallowed cellulose (PASC), the kinetics of the reaction, the processivity and the synergism with Tr. Cel7A. We characterised the wild type (WT) and 19 mutants of Tr. Cel5A CD absorption by using the substrate PASC prepared from Avicel. The absorption constant Kr with the values ranged from 0.12 to 0.39 L/g is obtained for the mutants, compared to that of the wild type (0.26 L/g). Several residues, including T18, K26, Y26, H229 and T300, are demostrated to be important for adsorption of the enzyme to PASC. High activity-low capacity adsorption is observed, indicating that the binding site is more accessible in some part of the substrate. The mutants also change the enzyme activity, which is probably caused by the disturbance of the enzyme active site structure, although these mutants are located at the binding surface of Tr. Cel5A CD. The experimental results show that there is no strong correlation of the enzyme activity and the adsorption of Tr. Cel5A CD to PASC. As the Tr. Cel5A CD catalyzed PASC hydrolysis progresses, the reaction slows down and the extent of the slowdown differs in all mutants and the wild type. The kinetic data show that the initial reaction rate A is negatively correlated with the reaction retardation power factor b values, which means that the faster initial reaction results in a quicker drop of the activity in the later stage. The reasons caused the slowdown may come from some obstacles in the substrate cellulose or the substrate heterogeneity that lead to different reactivity. The fitting result shows that the correlation between Kr and b is poor. A positive correlation is observed between the initial reaction rate of Tr.Cel5A CD against PASC and the specific activity of Tr.Cel5A CD against PNPC. The activity of Tr.Cel5A CD in hydrolyzing the soluble substrate appears to affect the initial reaction rate of PASC hydrolysis in some way. In the processivity assay, the WT and all the tested mutants show some degree of processivity. Among these, Y28A and W185A exhibit stronger processivity than the WT, whereas others show weaker processivity. The variants with different processivity also show different activities against PASC. The hydrolysis of PASC has been greatly improved when Tr. Cel7A is added into the reaction system, The strongest synergy presents in a Tr.Cel5A CD versus Tr. Cel7A ratio of 3:7. The processivity of Tr. Cel5A CD is negatively correlated with the synergism, meaning that the mutants with stonger processivity appear to have a weaker synergistic effect. Finally, by adding Tr. Cel7A in the reaction of Tr.Cel5A CD and PASC, the produced reducing sugar concentration differences between all mutants and WT decrease.
语种	中文
学科主题	仿真模拟
公开日期	2015-07-01
内容类型	学位论文
源URL	[http://ir.qibebt.ac.cn/handle/337004/8079]
专题	青岛生物能源与过程研究所_仿真模拟团队
作者单位	中国科学院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	舒志愚. 里氏木霉内切纤维素酶Cel5A催化机理研究[D]. 北京. 中国科学院研究生院. 2015.

个性服务

查看访问统计

相关权益政策

暂无数据

收藏/分享

除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。