多功能辅酶高效降解纤维素的协同作用机理研究

Cellulosic ethanol can be used as alternative to gasoline for vehicle transportation. One of the bottlenecks that hampers the commercial development of bio-ethanol is slow rate of enzymatic hydrolysis. Our previous study showed that a multi-functional enzyme (ME) StCel5A can enhance hydrolysis rate more than 25%. However, the synergistic mechanism of StCel5A is not elucidated yet. A central hypothesis of this research is that StCel5A can reduce crowding of cellobiohydrolase1 (CBH1) during the hydrolysis. The key element of this research work is to develop high-resolution liquid phase atomic force microscope (AFM) imaging for visualization and quantification of enzyme crowding. Active sites of StCel5A will be revealed using structural modeling and molecular docking. Activities of enzymes similar to StCel5A will be studied to further confirm the active sites. These works will provide molecular-level understanding of the synergistic mechanism of ME with CBH1. At the meantime, a new approach based on the critical amount of ME will be developed to evaluate the best synergism between ME and CBH1. The most efficient ME will be screened out to rationally design advanced enzyme system with CBH1. If successful, the fundamental knowledge gained in the proposed work will lead to new strategies for development of next-generation enzyme formulation, which is capable to increase efficiency of cellulose hydrolysis and decrease enzyme cost, allowing practical economic processing of cellulosic biofuel.

通过酶水解纤维素制取的生物质乙醇可以替代石油，将成为未来的主要车用燃料。然而，受水解速率缓慢等瓶颈的制约，生物质乙醇在经济性上还无法和石油燃料竞争。申请者前期的研究结果证实，使用多功能辅酶StCel5A能提高纤维素水解速率25%以上，但高效多功能辅酶协同降解纤维素的作用机理还不甚清楚，我们推测StCel5A对纤维素水解速率的提高与其降低外切纤维素酶CBH1凝结失活的作用有关。因此，本项目拟发展AFM液相清晰成像技术对CBH1的凝结进行表征，并综合应用结构模拟、分子对接、生物信息学等方法对StCel5A这类多功能酶的活性位点规律进行研究，进而在分子水平上揭示多功能辅酶协同水解生物质的机理；同时，本项目还将建立多功能辅酶临界用量的方法评价多功能辅酶的最优协同作用，运用该评价方法筛选最优的多功能辅酶，优化构建多功能辅酶和CBH1的酶体系，为开发基于多功能辅酶的纤维素水解高效酶配方奠定理论基础。

生物质乙醇做为一种可再生燃料，将在未来的车用燃料中扮演重要角色。然而， 受水解速率缓慢等瓶颈的制约，生物质乙醇在经济性上还无法和石油燃料竞争。使用多功能辅酶能极大提高纤维素水解速率，但在此研究之前，其作用机理还不甚清楚。通过该研究，我们发现，多功能辅酶的作用与酶的木聚糖活性高度相关。通过综合应用结构模拟、生物信息学等方法对StCel5A这类多功能酶的活性位点规律进行研究，发现能发挥功效的多功能辅酶具有除原有活性位点之外另一对谷氨酸活性位点，而不具有木聚糖活性的酶则都没有该活性位点，从而揭示了多功能辅酶协同水解生物质的机理；同时，本项目还建立了多功能辅酶评价方法，为通过蛋白工程改造多功能辅酶，开发基于多功能辅酶的纤维素水解高效酶配方奠定理论基础。