嗜热菌C.bescii高丰量非酶结构域在高效水解天然纤维素中的功能分析

Cellulase’s consumption and complicated pretreatment process are the bottleneck of lignocellulosic biomass to bioethanol production. Caldicellulosiruptor bescii is the most widely researched extremely thermophilic bacterium with the best efficiency in hydrolysis of cellulose so far described. To our surprise, C. bescii secretes high abundant non enzyme domains accompanying with several extremely thermophilic difunctional glycoside hydrolases (di-GHs). The function of non enzyme domains has not been reported. Therefore, this project plant to.heterogeneously express non enzyme domain Athe_0597 and di-GHs to investigate the function of Athe_0597.In order to accomplish above, fluorescent labeling and observation of localization, hydrolysis efficiency, substrate structural change and so on will be carried out to study the synergy and mechanism of Athe_0597 to di-GHs degrading lignocellulosic biomass. In addition, molecular modification of Athe_0597 will be performed through rational design and site-directed mutagenesis. The mutant will be constructed to verify the synergic mechanism of Athe_0597. Research results is expected to be applied to a new process of lignocellulosic bioethanol, to omit complicated pretreatment, decrease environmental pollution and reduce production cost.

纤维素酶成本和复杂预处理工艺是纤维素乙醇商业化生产的瓶颈。Caldicellulosiruptor bescii是目前研究最为广泛的水解天然纤维素能力最强的嗜热细菌，能够分泌高效水解天然结晶纤维素多酶体系。非常有趣的现象是C.bescii分泌多种极耐热双功能糖苷水解酶（di-GHs）时相伴而生高丰量非酶结构域，关于C.bescii非酶结构域功能未见报道。本项目拟以C.bescii非酶结构域Athe_0597为研究对象，针对其功能，将Athe_0597和di-GHs异源表达，通过荧光标记和分布观察、水解效果测定、底物结构变化观察等，研究Athe_0597对di-GHs水解结晶纤维素的协同作用和作用机制；通过理性设计、定点突变的技术手段进行蛋白质分子改造，构建Athe_0597突变体，验证Athe_0597的功能。研究结果有望应用于纤维素乙醇新工艺，省去复杂预处理，减少环境污染，降低生产成本。

嗜热菌Caldicellulosiruptor bescii能够高效水解天然结晶纤维素。在C.bescii分泌的高效降解纤维素体系中，有些非酶结构域与糖苷水解酶相伴而生，而且分泌量丰富，其功能尚未明确。本项目选择两种高丰量非酶结构域Athe_0597和Athe_0181为研究对象，探索Athe_0597和Athe_0181在C.bescii高效水解结晶纤维素中的功能。研究发现Athe_0597和Athe_0181具有对C.bescii双功能糖苷水解酶Athe_1866水解微晶纤维素、水稻秸秆、棕榈粕的协同促进功能。对协同水解微晶纤维素的作用条件进行了探索，在两种非酶结构域作用下，Athe_1866水解微晶纤维素12h所产生的还原糖量分别是未添加Athe_0597和Athe_0181的2.06倍和1.95倍。对两种非酶结构域的协同机理进行了探究，研究发现Athe_0597和Athe_0181对多种纤维素、半纤维素没有水解能力，与微晶纤维素、水稻秸秆、棕榈粕结合能力较强。采用SEM、FTIR、XRD等方法对Athe_0597和Athe_0181作用后的底物结构进行分析观察，发现两种非酶结构域对微晶纤维素底物结构具有一定的改变作用。研究结果为纤维素乙醇和生物化学品的制备新工艺提供重要的理论和技术基础。