极端微生物纤维素酶TmCel12A耐超高温特性机理研究

Cellulase, an enzyme that hydrolyses cellulose into glucoses, has great potential for renewable energy, such as biomass energy. However, the low efficiency, poor thermal stability and the end-product inhibition effect become the bottleneck for the application of cellulases. Today, with energy crisis becoming more and more severe, researches that focus on improving the activities and thermostabilities of cellulases attract more and more attention. Recently, the crystal structure of a cellulase, TmCel12A, with an optimum temperature of 85°C has been solved and the newly found gain-of-function mutation, Y61G, has been proved to improve both the hyperthermal stability and reaction activity of TmCel12A. These experimental achievements provide a solid base for further study of the hyperthermal nature of TmCel12A. Based on our research experience and the preliminary results, we proposed to clarify the mechanism of the hyperthermal stability of TmCel12A from the aspects of the enzyme thermal stability, the thermal stability of enzyme-substrate complex, the formation of the enzyme-substrate complex, the release of the products and relationship between the thermal stability and the activities of TmCel12A. A combination of both theoretical and experimental approach including molecular dynamics simulation, quantum mechanics, QM/MM, free energy calculation, enzyme activity determination, and enzyme dynamics profile measurement will be applied. It will provide a theoretical guide for the improvement modification of not only cellulases but also other hydrolysis enzymes, therefore of great help in promoting the massively application of biomass energies.

纤维素酶能将纤维素水解成葡萄糖，在生物质能源的开发利用中具有巨大的应用潜力。然而它催化效率低、热稳定性差等缺点成为制约其应用的瓶颈。在能源危机日益严重的今天，改良纤维素酶催化活性、提高其热稳定性的研究受到广泛关注。最近，具有85°C最适反应温度的纤维素酶TmCel12A晶体结构的获得和具有进一步提高其耐超高温特性的Y61G突变体的发现为研究TmCel12A耐超高温特性的机理提供了可靠的实验基础。本项目拟以此为突破口，在前期工作基础上，利用理论与实验相结合的方法综合运用分子动力学模拟、量子力学-分子力学多尺度模型、自由能计算以及酶活性测定、酶反应动力学参数测定等方法，从酶自身热稳定性、酶-底物复合体热稳定性、复合体形成和产物释放、以及热稳定性与催化活性相关性等四个方面全方位研究TmCel12A耐超高温特性的机理，为设计改良其它纤维素酶及类似水解酶提供可靠的理论基础，推动生物质能源的开发利用。

本项目按计划任务书，针对纤维素酶TmCel12A耐超高温的特性，设计了一系列的理论计算实验，较好的完成了计划任务，取得的主要研究结果如下：1.首次发现了纤维素酶TmCel12A空酶在高温情况下形成以Y61和W176为核心的稳定的疏水中心的现象，以此为出发点，通过一系列模拟计算和分析解释了Y61在调节纤维素酶TmCel12A催化活性和耐超高温特性中的作用机理；2.提出了功能获得型突变Y61G通过改变纤维素酶TmCel12A内部氨基酸动态相关性网络结构，增加氨基酸之间长程相互作用，增强纤维素酶TmCel12A柔性从而提高了其高温稳定性的机制；3.发现了 Y61G突变消除了纤维素酶TmCel12A通过Y61侧链与底物+2位葡萄糖之间的π-π相互作用，改变了反应产物从酶活性口袋中的释放的过程，从而提高了纤维素酶TmCel12A在高温环境中的催化速率；4. 对5-HT1A受体激动剂的计算机辅助药物设计中，首次提出了药物分子在结合口袋中的多态性，验证并利用该特性设计了一系列具有较高活性和选择性的潜在5-HT1A受体激动剂分子。本研究中对纤维素酶TmCel12A耐超高温机理的发现对于改造已有纤维素酶提高其热稳定性从而推动我国清洁能源的开发利用具有重要的参考应用价值。