里氏木霉外切纤维素酶CBH I的水解机理研究和理性设计

Enzymatic cellulose hydrolysis is studied intensively due to the potential applicability in production of sustainable bioethanol and chemicals. The cellulases from Trichoderma reesei, which are expressed with high yield, receive much attention. In order to decrease the enzyme cost in production, significant efforts have been expended to understand the mechanisms and improve the activities of natural cellulases. However, the crucial enzymes such as Cellobiohydrolase I (CBH I) work at solid−liquid interface. It is difficult to reveal the detail experimentally, and the mechanisms are still not clear. Combining the molecular dynamics simulation, the molecular biology and enzymatic experiment, the crucial hydrolysis processes of CBH I will be investigated in this project, including the adsorption on cellulose surface, the cleavage of glycosidic bond and the processive motion. Some mutations that may regulate these progresses are first designed by theoretical calculation, and their impact on enzyme adsorption, cleavage, processive motion and activity against various substrates will be evaluated in experiment. By analyzing the correlation between each progress and total activity, the rate-limiting step would be determined. Based on the experimental data, the structure factors that regulate hydrolysis progresses are further studied by simulation, and more mutations will be introduced through computer-aided design to improve the enzyme activity. The combined study will help to elucidate the catalytic mechanism of CBH I and promote its application in lignocellulose conversion. In addition, the method employed in this project will offer hints to other studies of enzyme catalysis occuring on solid-liquid surface.

纤维素酶水解纤维素为单糖，是木质纤维素利用中最受关注的生物转化途径之一。然而，纤维素的降解发生在固液两相表面，传统酶学方法很难用来研究该过程。本项目将以里氏木霉外切纤维素酶CBH I为研究对象，结合分子动力学模拟和分子生物学/酶学实验，阐明酶水解纤维素过程中的吸附、剪切、滑移机制，揭示各步骤与水解活性的相关性，进而通过理性设计提高CBH I的活性。本项目的顺利实施将有助于提升里氏木霉纤维素酶系的催化效率，同时也为其它固液两相的酶催化反应研究提供新思路。

本项目首先以持续性内切纤维素酶CcCel9A为研究对象，结合计算模拟与实验，解析了其多个底物结合模块及活性中心关键位点的功能，提出了多模块持续性内切纤维素酶降解结晶底物的新模式—“wire-walking”模式，本工作为纤维素酶性能改进提供了新的设计思路。此外，理性设计的精度依赖于对蛋白质结构与功能关系的理解程度，特别是非共价键相互作用的认知程度。在本项目的资助下，我们开展了蛋白质静电作用和范德华力的相关研究。静电作用发面，我们发展了高精度的核磁共振盐桥检测方法，首次实验揭示了GB3蛋白表面盐桥的熵驱动形成机制，进一步的计算模拟解析表明这种驱动力主要源于溶剂水的熵效应。范德华力方面，我们发展了一种新的核磁共振检测方法，首次实现了GB3蛋白体系跨范德华力的JCC-耦合常数的实验测量(数值在~0.1-0.6 Hz之间)，进一步的理论计算结果表明跨范德华力的JCC-耦合常数与范德华力的交换-互斥力组分正相关。最后，在本项目的资助下，我们继续发展了可定量描述蛋白质内电子信号传递速率的键耦合模型，通过不同长度、不同二级结构多肽链的对比研究，解析了蛋白质构象变化中影响其双向传递速率的关键要素。这些工作的开展为理解、分析进而调控蛋白质功能提供了新思路。