极端嗜热细菌Caldicellulosiruptor中双功能纤维素水解酶作用机制的研究

Conversion Plant-derived biomass to fuel alcohols must first break down lignocelluloses into component sugars. Many glycoside-hydrolytic enzymes, involved in this process, were extensively studied, including Cellulosome from Clostridium, or free glycoside hydrolases (GH) secreted from Trichoderma. In this research proposal, we will investigate a series of unique multidomain Bifunctional Lingo-Cellulases (BLCases), which are composed of GH families 5, 9, 10, 44, or 48, and family 3 Carbohydrate-Binding Modules (CBM3) from the extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor. Base on achievement of previous results and our preliminary work, we will use the strategies of, hetero-expression of BLCases, deletion (or replacement) of functional modules, 3D structural prediction of BLCases or their modules, analysis of catalytic-active sites/carbohydrare-binding sites, analysis of substrate specificity and etc., to clarify the relationships of each module in BLCases (XylA, CelB, and XGlA) and their roles in straw degradation. Meanwhile, we will produce the highly-specific mouse poly-antibodies towards BLCases to verify if other unknown factors (such as other BLCases or Surface-layer proteins) are involve in BLCase-participant straw degradation using the technologies of Co-immunoprecipitation (Co-IP) and His Pull-down assay. Taken together, this project could improve our understanding of the straw degradation by BLCases, and give a theoretical basis to design the high-quality BLCases.

在生物质原料-秸秆转化成醇类燃料的过程中，首先需要将秸秆中的复杂多糖分解为单糖。许多参与此过程的糖苷水解酶已被广泛研究，如梭菌属的纤维小体和木霉菌属的游离型水解酶。在本项目中，我们将研究一类独特的、带有多个结构域的、双功能木质纤维素水解酶（双功能酶）。源自纤维素热解杆菌双功能酶是由两个催化模块和2-3个底物结合模块组成。我们将以前人研究结果和前期工作为基础，使用蛋白异源表达、模块缺失删除或替换、蛋白3D结构预测、催化活性和底物结合位点分析、底物特性分析等多种策略，阐明双功能酶 （XylA、CelB、XGlA） 中各模块之间的关系，以及双功能酶在降解秸秆过程中的作用。同时将制备针对双功能酶的高特异性小鼠多抗，通过免疫共沉降、His-沉降等方法探寻是否有其它未知因子协同双功能酶参与木质纤维素糖化过程。最终通过本项目，加深对双功能机制的理解，并为设计和开发高质量的双功能酶提供理论依据。

本项目以热解纤维素梭菌Caldicellulosiruptor为研究对象，通过基因组测序技术获得了一个Caldicellulosiruptor菌株的基因组草图。通过生物信息学进行注释和分析，获得了该菌株中双功能型的木质纤维素降解酶及其相关辅助酶系。随后利用大肠杆菌蛋白重组表达体系、蛋白纯化、酶学及生物化学表征、毕赤酵母高效表达等多种技术手段，获得双功能性纤维素降解酶以及相关的木质纤维素降解酶系。取得的成果（1）本项目组分离出一株Caldicellulosiruptor菌株，通过测序和表征，命名为C. sp. F32，通过基因组测序，分析了其中双功能型木质纤维素水解酶的类别和分布，为功能基因的挖掘利用创造基础条件；成果（2）获得了获得的双功能型阿拉伯呋喃糖苷水解酶XynF与内切型木聚糖酶XynA进行复配，可将阿拉伯木聚糖的水解产物的得率与对照相比提高了6倍以上。成果（3）在C. sp. F32菌株中发现两个多模块的内切性木聚糖酶（GH11家族的XynA和GH10家族的XynB），这两个酶与双功能型阿拉伯呋喃糖苷水解酶XynF复配时，均有较好的协同能力。这些结果都为下一步酶制剂的商业化应用提供可行性方向。