宏基因组库中新双功能木聚糖酶的富集后筛选及其定向改造

The degradation of xylan and other natural polysaccharides needs collaboration of multiple enzymes. The bifunctional xylanases have obviously lower costs and higher catalytic efficiency when compared with the mixtures of single-functional enzymes. However, now there are only very few known bifunctional xylanases which have unsatisfactory activities and thermal stabilities. Additionally, it is inefficient to obtain new bifunctional xylanases by using the commonly used function-based metagenomic library screening method. In this study, a Recombination Protein A-based method will be used to enrich bifunctional xylanase genes, and new bifunctional xylanases would be identified by screening the enriched library. Then some new bifunctional xylanases should be chosen to construct DNA shuffling and incremental truncation libraries, followed by reading frame selection and function screening of the libraries to obtain mutants with improved activities and thermal stabilities. Finally, enzymatic and bioinformatics characterizations of the bifunctional xylanases before and after directed evolution will be studied, which should lay the foundation for future study and application of these enzymes.This study should be an important improvement of the current metagenomic techniques, which can significantly increase copy numbers of target genes, and increase the chance of obtaining target proteins. In addition, the screening platform established in this study would lay the foundation for obtaining other novel bifunctional xylanases in the future.

木聚糖等天然聚糖的降解需要多种酶的协作作用。而相对于单功能酶的混合物，双功能木聚糖酶在降低成本、提高催化效率等方面都具有不可比拟的优势。但目前已知的双功能木聚糖酶数量很少，并且在活性和热稳定性等方面不够理想，而采用目前常用的宏基因组库技术结合功能筛选获得新双功能木聚糖酶的效率很低。本研究将利用一种基于重组蛋白A的方法先对宏基因组库中含目的基因的片段进行富集，并在此基础上筛选获得新的双功能木聚糖酶。然后构建DNA shuffling库和渐进性截短库，再经过读码框选择和功能筛选，进一步提高酶的活性和热稳定性。最后对定向改造前后的酶进行酶学特征测定和生物信息学分析，从而为双功能木聚糖酶的应用及深入研究奠定基础。本研究将是对现有宏基因组技术的重要改进，可以显著提高目的基因的拷贝数，增加筛选获得目的基因的概率。同时，本研究所建立的筛选平台也将为未来更多新双功能木聚糖酶的筛选奠定基础。

木聚糖等天然聚糖的降解需要多种酶的协作作用。而相对于单功能酶的混合物，双功能木聚糖酶在降低成本、提高催化效率等方面都具有不可比拟的优势，在研究酶的进化规律方面也有重要的价值。但目前已知的双功能木聚糖酶数量很少，并且在活性和热稳定性等方面不够理想，从而严重制约着其理论研究和生产应用。本研究用含有木聚糖和阿魏酸乙酯的培养基富集培养微生物，然后构建宏基因组库，并在此基础上筛选获得了多个新的双功能木聚糖酶，在提高了酶的热稳定性和活性的同时，也在生物信息学水平上对酶的特征进行了分析，从而为双功能木聚糖酶的应用及深入研究奠定基础。在此基础上，还改进了宏基因组DNA提取方法和建库方法，对相关研究具有重要的参考价值。