纤维素多酶复合体降解木质纤维素的协同效应机制

The challenges for sustainable development in China are the depletion of energy and environmental pollution. Since lignocelluloses are the most abundant bioresouces in the earth, the utilization of cellulosic materials called as biorefinery becomes a very successful system to produce renewable energy. The biofuels and biorefinery product from lignocellulosic biomass are the alternative solutions for the impact of climate change, resources exhaustion and industrial evolution. However, the complex of lignocelluloses, i.e. the compositions, structures and sources caused the difficulty in degradation. As a result, the mechanism and utilization of lignocelluloses are still unclear. This research based on the biotechnology is first attempt to establish the platform for high-through put screening of novel bacteria with potential cellulases via zymogram and proteomics approach to prevent the patent protection by international companies. Novel genes further improved by site-directed mutagenesis and cloned into E. coli to accomplish the over-expressed enzyme system. The analysis of morphology, chemical composition of lignocelluloses through the modern analytical technologies, including Near Infrared Spectroscopy (NIR), Scanning Electron Microscopy (SEM) and X-ray provided the database and information to understand the basic chemical and physical properties of the materials. The study on synergistic mechanism of cellulases and molecular interactions between enzymes are effectively for the cellulases formulation. This task reveals the theory for biodegradation of lignocelluloses to adapt in biorefinery application.

能源短缺和环境污染是我国持续发展面临的两大挑战。木质纤维素是地球上蕴藏量最丰富的生物质资源，以木质纤维素进行生物炼制达成可持续发展的能源系统，对环境、能源、工业改革等难题具有重大意义和影响。但因为木质纤维素的来源广泛，其组成和结构极不相同，影响降解因素错综复杂，导致其利用及机理研究并不透彻。本项目采用新一代生物技术路线，以酶谱法串联蛋白质组学分析，建立高通量纤维素酶筛选平台挖掘新颖菌株，突破国际大厂的专利保护抵触；藉由定向进化技术改造基因并完成其重组大肠杆菌外源表达系统；同步建立木质纤维素组成和结构的数据库；采取现代分析技术近红外线光谱(NIR)、扫瞄式电子显微镜(SEM)与X-射线(X-ray)等方法侦测木质维素素材料的型变及质变，阐明木质纤维素的物化性质，揭示纤维素酶其协同效应的降解机制，弄清酶分子间的相互作用，开发有效的纤维素酶制，为纤维素酶在木质纤维素的生物炼制提供理论依据。

提升生物质资源的利用效率，能够有效解决人类面临的能源短缺问题和环境污染问题。木质纤维素是地球上蕴藏量最大的生物质资源之一，研究表明，利用纤维素酶水解纤维素是木质纤维素利用的关键技术。纤维素酶的来源广泛，目前多采用真菌如里氏木霉来生产，但是其受到国际大厂的专利保护，因此突破其专利保护，筛选发掘新颖高酶活纤维素酶生产菌株具有很大的应用价值，本研究采用新一代生物技术路线，以酶谱法串联蛋白质组学分析，建立高通量纤维素酶筛选平台，并挖掘得到一株新颖菌株Klebsiella sp. YS2-1。将其纤维素酶基因cel8A重组表达于大肠杆菌中，酶活达到62.4 U/mg（基于CMC底物）。纤维素的酶解较为复杂，导致其利用及机理研究并不透彻，本项目通过NIR, PSD. SEM等手段测定了茶叶废弃物经里氏木霉菌纤维素水解酶处理前后的组成和结构，并发现处理后的农业废弃茶叶，可应用于吸附重金属Cr(VI)，并建立其动力学吸附模型，此研究对于吸附水溶液中的Cr(VI)和扩大农业废弃物的使用具有积极作用。同时，为了促进大肠杆菌表达系统生产纤维素酶的工业应用，课题通过裂解基因lysis融合纤维素酶基因构建成裂解回路来解决重组纤维素酶的破壁释放问题，已成功应用于基因cel8A的同步释放。这对于纤维素酶的大规模快速生产具有积极的促进作用。