液氨解构甜菜渣细胞壁及促进纤维素高效酶解的机制研究

Sugar beet pulp has been deemed as an ideal cellulosic material for fuel ethanol production. However, the low efficiency in enzymatic hydrolysis hinders the development of fuel ethanol production. Cognition of the critical recalcitrant barriers, selective removal of the recalcitrant barriers and destruction of cell wall become the vital subject in the enzymatic hydrolysis. Our previous results showed that the aqueous ammonia pretreatment degraded the pectin of sugar beet pulp, led to cell wall dislocation and lignin redistribution, destroyed rigid structure and enhanced enzymatic hydrolysis. However, the interactive effects of chemical compositions and physical structures, and their mechanism on enzymatic hydrolysis are still ambiguous and should be studied further more. In this project, various advanced analytical methods will be used to verify the recalcitrant factors in enzymatic hydrolysis and elucidate the mechanism of aqueous ammonia pretreatment in the aspects of macrostructure, microstructure, ultrastructure, molecule, chemical group, chemical element, and chemical bonds level. Firstly, the influence of aqueous ammonia pretreatment on the chemical composition content, migration and distribution in the cell wall, and the morphology and structure of the cell wall will be analyzed. Furthermore, the relationship between the various physicochemical factors and the enzymatic hydrolysis efficiency will be discussed to uncover the recalcitrant barriers and their particularity. Finally, the mechanism of aqueous ammonia pretreatment will be clarified based on the variation of recalcitrant barriers. This project will uncover the recalcitrant barriers of sugar beet pulp, and clarify the dissolution and redistribution of various compositions in sugar beet pulp during the aqueous ammonia pretreatment. Additionally, the effect of chemical variation on the physical destruction of cell wall structure will also be analyzed. Not only is the related material representative and region characteristic, but also the research strategy is unique. The results of this project will enrich the theory of enzymatic hydrolysis of cellulose, and lay the theoretical and technical basis for the utilization of sugar beet pulp.

甜菜渣是制备纤维素燃料乙醇的理想原料，但是纤维素酶解效率低制约着其产业化发展。从本征的理化结构出发，剖析甜菜渣的抗降解屏障，选择性破坏关键顽抗因子，解构细胞壁，是实现甜菜渣纤维素高效酶解的关键。实验室已证实液氨能够选择性去除果胶、引起木质素再分布，提高酶解效率，但是液氨预处理过程中甜菜渣细胞壁组分的溶出及迁移的规律，化学组分的变化和细胞壁结构变化间的联系，及二者变化对甜菜渣纤维素酶解效率的影响仍不明确。本项目拟采用多种先进分析技术从甜菜渣宏观形态、显微结构、超微结构、分子、基团、元素、化学键等水平展开研究。首先，研究液氨预处理对甜菜渣化学组成及分布、物理结构及形态的影响；通过分析细胞壁理化性质变化与纤维素酶解效率的关系揭示甜菜渣的抗降解屏障及其特殊性；通过探讨抗降解屏障的变化阐明液氨预处理促进甜菜渣纤维素高效酶解的机制。项目的实施将揭示甜菜渣的抗降解屏障，阐明液氨预处理过程中甜菜渣的不同组分溶出、再分布规律，及其对细胞壁物理形态的解构机制。研究材料具有代表性和主体地域特色，研究策略独特。预期研究成果将丰富纤维素酶催化反应理论内涵，为甜菜渣高值化利用提供重要的理论基础与技术储备。

本项目采用了多种先进分析技术，全维度地考察了液氨预处理对甜菜渣细胞壁化学组成、组分分布、物理性质、形态结构和酶解效率的影响，以期揭示甜菜渣的抗降解屏障及其特殊性，并初步阐明液氨预处理作用机制。主要研究结论如下:（1）建立了细胞壁数字化分析法方法和结合荧光蛋白的纤维素酶探针分子，为从细胞壁水平探究液氨预处理对纤维素的作用机制提供了技术支持。（2）证实了液氨预处理引起甜菜渣木质素的再分布与局部聚集，弱化了纤维素与木质素间的相互作用，暴露纤维素结晶区，显著提高了纤维素酶最大吸附量、酶解初速率和酶解还原糖得率。（3）明确了甜菜果胶、半纤维素和木质素之间存在较多的交联，半纤维素Non-FUC XG聚糖最为顽固，果胶主链比支链与细胞壁结合更为牢固；AG-3、RG、HG Backbone和木葡聚糖与还原糖得率呈显著负相关，是制约甜菜渣纤维素酶促水解反应效率的关键聚糖；甜菜的薄壁组织更容易与纤维素酶结合，且更易被降解，而细胞壁角隅最难被酶降解。（4）液氨预处理过程中，不同聚糖的溶出和分布规律不同。RG-ⅠB和RG-ⅠC聚糖在0.5 h内几乎全部溶出，FUC XG、Non-FUC XG和HG Backbone聚糖前3 h内大量溶出、后趋缓，AG类聚糖逐渐溶出，Xylan类聚糖较难溶出；M 23、M 131和M 145识别的聚糖在细胞角隅处含量更高，且液氨预处理后M131和M145识别的聚糖会聚集在细胞壁角隅处。（5）液氨预处理能够降解柠条半纤维素和中性洗涤物，保留纤维素；显著溶出果胶/阿拉伯半乳聚糖等细胞壁非纤维素聚糖，断裂柠条木质素与细胞壁聚糖交联结构中的酯键、醚键和氢键；破坏柠条致密结构和细胞壁的完整性，增大孔隙率，暴露纤维素结晶区，增加可及度。（6）液氨预处理甜菜渣的最适反应条件：氨水浓度10%（w/w）、固液比1:10、80℃、静置、3 h；甜菜渣的纤维素酶促水解反应条件：20 FPU/g、固液比1:20、50℃、振荡36 h；甜菜渣纤维素酶解还原糖得率达到437.7 mg/g，是对照组1.87倍。该项目丰富了纤维素酶促水解理论，为甜菜渣高值化利用提供重要的理论基础与技术储备。