糖苷水解酶GH5亚家族活性中心架构及其功能快速演化的相关机理研究

In the new century, along with the change of cropping system in China , the efficient utilization of straw resource has become a prominent problem of social development. Rational designing and constructing efficient glycosidase system has been considered as the key to solve these problems. In this project, the genetic diversity and different distributions of GH5 subfamilty have been explored using metagenomics technology. With the construction of phylogenetic relationships among sequences and analysis of catalytic active architecture functional areas among different subfamilies, we can predict their covariability of residues/combination in fast evolution. Based on theoretical calculation analysis, we determine the properties and function of specificity determining residues/combination, clone related templates and mutant the related residues. Additionally, through the dynamic spectrum technology, micro thermal technology and fluorophore-assisted carbohydrate electrophoresis (FACE), tracking and designing the reaction kinetics process of new enzymes revealed the new active architecture and the mechanisms of the fast function evolution of GH5 subfamily. This study is also helpful for the accuracy of metagenomic sequence function annotation and the success rate of protein engineering redesign. These results will provide the theoretical foundation to locate the composition and function of key enzyme components in high efficient biomass conversion systems, discover and design new enzyme components and new protein factors purposefully, and construct high efficient lignocellulose degrading enzymes for biorefinery industry.

进入新世纪，随着我国农作模式的转变，秸秆资源的高效利用已成为社会发展突出问题，理性设计并构建高效糖苷酶系成为解决该问题的关键。本课题利用宏基因组学技术研究GH5亚家族序列的多样性与分布丰度；通过结构生物信息学方法构建序列之间的聚类关系，分析不同亚家族的催化活性架构功能区，预测其快速演化的残基/组合及其协变性；利用理论计算分析决定功能专一残基/组合的性质与功能；克隆相关模板，针对相关残基进行重新设计；结合动态光谱技术、微量热技术、荧光辅助糖电泳技术等跟踪检测再设计新酶的反应动力学过程，在分子相互作用水平上认识GH5亚家族的活性架构及其功能快速演化的相关机理。该研究可提高宏基因组序列功能注释的准确度，提高蛋白质工程设计的成功率。相应成果的取得将为研究高效转化系统中定位关键酶组分的组成及功能、有目的发现并设计新酶组分及新蛋白因子，构建高效木质纤维素降解酶系统奠定理论基础。

我国进入新世纪以来，随着农作模式的转变，高效利用秸秆废弃物资源已成为促进发展的突出问题，通过理性设计方法构建高效糖苷水解酶系是解决该问题的关键。本课题利用整合宏组学技术研究了GH5等糖苷水解酶家族的序列多样性与分布丰度；通过结构生物信息学的方法构建了序列之间的聚类关系；建立分子动态组学方法，分析了不同家族与亚家族酶分子的活性功能区分子动态行为，从而发现GH5不同亚家族远端（-2与-3亚位点）是功能快速演化区域；同时利用分子动态组学方法分析了决定功能专一残基/组合的性质与功能；利用湿实验克隆了两种GH5家族的酶分子TfCel5A、TrCel5A，并对其活性架构进行丙氨酸筛选及功能残基的重新设计；运用荧光辅助糖电泳技术、荧光光谱技术、结合力电泳技术和等温滴定量热技术跟踪分析了酶分子的反应动力学过程，在分子水平上认识GH5亚家族功能快速演化区域及结合催化的相关机理。并且，相关研究已经进一步拓展到GH7、GH11及GH12等糖苷水解酶家族，从而使蛋白质理性设计的概率得到明显提高。本课题所获得的研究成果将为定位和研究高效转化系统中的关键酶组分及其功能，发现并设计新酶组分及新蛋白因子，构建高效木质纤维素降解酶系奠定理论基础。