一组高效纤维素降解复合菌系的关键降解因子及其作用机制研究

The large-scale exploration and utilization of waste agro-industrial lignocellulosic resources, which has abroad distribution and huge output, is contrainted for the lack of cheap and effective pretreatment technology, thus resulted in great environmental-pollution and tremendous resource-wasting. In this study, the efficient transformation of the abandoned lignocellulosic resources into biogas is focused as the research guidance. On the basis of the directional constructed microbial consortium with high efficient cellulolytic ability in our prior studies, this project tries to find out the dilution critical point for the effective cellulose degradation by the microbial consortium using serial dilution method. Furthermore, combined with the technology of environmental genomics, the key functional microbes for cellulose degradation are clarified and their degradation mechanism will be further elucidated. In addition, based on the serial dilution, affinity digestion is utilized for the extraction and purification of the cellulose-binding proteins of this microbial consortium. Moreover, combined with the technology of metaproteomics, western blot, immunohistochemistry and enzyme spectrum analysis, the composition, excretion, expression and the distribution on the substrates of these functional proteins produced by this microbial consortium are studied, and their action mechanism is analyzed. Finally, the high efficient cellulolytic mechanism of this microbial consortium is elucidated from the angles of function microbe and zymoprotein respectively, which would further provide certain theoretical basis and technical support for the efficient biogas transformation of lignocellulosic resources.

工农业废弃的纤维质资源分布广泛，产量巨大，因缺乏便宜、有效的处理技术，限制了其大规模的开发利用，从而对环境造成了极大的污染并导致资源的巨大浪费。本研究以废弃纤维质资源的高效沼气转化为研究导向，在前期定向构建的一组高效纤维素降解复合菌系的研究基础上，本项目采用梯度稀释法找到此复合菌系有效降解纤维素的稀释临界点，再结合环境基因组学技术，弄清纤维素降解的关键功能菌并阐明其作用机制；此外，在梯度稀释的基础上，采用亲和消化法提取纯化复合菌系中的纤维结合蛋白，再结合环境蛋白组学、蛋白质印迹、免疫组织化学和酶谱分析技术研究复合菌系中功能蛋白的组成、分泌表达及其在降解底物上的分布并分析其作用机制；最终分别从功能微生物和酶蛋白的角度阐述此复合菌系的纤维素高效降解机制，为纤维质资源的高效沼气转化提供一定的理论依据和技术支撑。

工农业废弃的纤维质资源分布广泛，产量巨大，因缺乏便宜、有效的处理技术，限制了其大规模的开发利用，从而对环境造成了巨大的污染并导致资源的极大浪费。本研究以废弃纤维质资源的高效沼气转化为研究导向，在前期定向构建的一组高效纤维素降解复合菌系的研究基础上，采用梯度稀释法确定了复合菌系有效降解纤维素的稀释临界点为10-5，再结合环境基因组学技术，确定菌株Clostridium clariflavum DSM 19732和Paenibacillus sp.是复合菌系中的关键降解功能菌。此外，在梯度稀释的基础上，采用亲和消化法提取纯化复合菌系中的纤维结合蛋白，再结合环境蛋白组学技术和酶谱分析技术，鉴定出了8种来源于菌株C. clariflavum DSM 19732和Paenibacillus sp.的纤维结合蛋白，且发现纤维结合蛋白CBP6(97.2kDa)和CBP12(50kDa)在复合菌系的高效降解中起着最为关键的作用。结构域分析和酶活性检测发现，CBP6和CBP12分别拥有第9和48糖苷水解酶家族的结构域，它们同时分泌的内切纤维素酶与木聚糖酶可以协同促进纤维素的降解。随后，对纤维结合蛋白CBP6和CBP12分别进行了分离纯化及抗体制备，再通过免疫组化和免疫荧光技术分析发现蛋白CBP6和CBP12在复合菌系的高效降解过程中会紧紧地附着在纤维质的表面上，从而促进纤维质的高效分解。此外，还研究了纤维素降解复合菌系对纤维质资源厌氧发酵的生物强化技术，发现纤维素降解复合菌系可以显著提高厌氧消化效率及厌氧消化的稳定性。上述研究成果为纤维素降解复合菌系的大规模应用及纤维质资源的高效沼气转化提供一定的理论依据和技术支撑。