基于纳米力学分析木质素与纤维素酶间的离子和非离子-π作用机制

After pretreatment, residual lignin can produce serious non-productive adsorption to cellulase, which significantly increases the production cost of cellulosic fuel ethanol by 30% to 50%. Based on the interaction mechanism between lignin and cellulase, shielding lignin or regulating cellulase structure will be the fundamental way to solve this problem. However, previous studies only limited to electrostatic, conventional hydrophobic and hydrogen-bonding interactions between lignin and cellulase, restricting the directional development of efficient lignin shielding agent and the precise regulation of enzyme molecular structure. In view of these questions, the functional groups that initiate the π electron-based interactions will be firstly identified in this project based on the nanomechanical analysis. Besides, the surface force apparatus and density functional theory will be used to analyze the adsorption force and adhesion energy between lignin and cellulase, and therefore reveal their interaction mechanism of ion-π (such as cation-π and anion-π). Moreover, the contributions of alkyl and aryl, aliphatic and phenolic hydroxyl groups to the hydrophobic and hydrogen-bonding interactions will be comparatively analyzed, respectively. These results will be beneficial to elucidate the strengthening mechanism of the non-ion-π (π-π stacking and p-π conjugation) effects on the hydrophobic and hydrogen-bonding interactions between these two macromolecules. Finally, according to the changes in the spatial conformations of catalytic and binding domains of cellulase components, the influencing mechanism of ion-π and non-ion-π interactions on the catalytic efficiencies of cellulase components will be explored. The implementation of this project will lay solid theoretical basis for enriching the non-covalent interaction system between lignin and cellulase, thereby promoting the industrialization of cellulosic fuel ethanol.

预处理后残余木质素可对纤维素酶产生严重的无效吸附，显著增加燃料乙醇的生产成本（30~50%）。根据木质素与纤维素酶的作用机制，屏蔽木质素或调控纤维素酶结构将是解决此问题的根本途径。然而，前期研究仅局限于两者间静电、常规疏水和氢键作用，限制了高效屏蔽剂的定向开发及酶分子结构的精确调控。据此，基于纳米力学分析，本项目首先确定引发π基作用力的官能团；其次，利用表面力仪和密度泛函理论分析两者间的吸附力和粘附能，揭示其离子-π（阳离子-π和阴离子-π）作用机制；同时，区分烷基、芳基及脂肪羟基、酚羟基对疏水和氢键作用的贡献，阐明非离子-π（π-π堆积和p-π共轭）效应对两者间疏水和氢键作用的强化机制；最终，根据酶组分催化和结合域空间构象的变化，探明离子-π和非离子-π作用对纤维素酶催化效率的影响机制。项目研究将为完善木质素与纤维素酶间的非共价作用体系奠定坚实理论基础，促进燃料乙醇的产业化发展。

木质素对纤维素酶的无效吸附是影响生物炼制经济性的重要影响因素，研究两者间相互作用是降低或消除此不利因素的理论基础。除静电和氢键等常见作用外，首先，本项目在液相缓冲体系中原位测定木质素-纤维素酶分子层间的离子-π作用，相应的黏附能（~2.89 mJ/m2）比纤维素-纤维素酶 （~ 1.48 mJ/m2）高95%，理论计算木质素模型物与阳离子间的结合能进一步验证此实验结果；其次，研究发现，模型分子（如聚N-乙烯基己内酰胺）与木质素间主要通过疏水作用产生吸附；同时，基于对不同来源预处理固体基质及木质素结构的表征，开发非水解植物蛋白（如大豆蛋白、花生蛋白和玉米胚芽蛋白等）作为生物基木质素屏蔽剂，有效降低纤维素酶在木质素表面的吸附量，将PSA预处理竹材和桉木等固体基质中葡聚糖的酶解转化率从大约40%提升至90%以上；最终，提出屏蔽剂经离子-π、静电、氢键和疏水等多种作用对木质素产生屏蔽的综合作用机制，为开发屏蔽剂以及提升木质生物质酶解效率提供了重要的理论根据。目前，本项目已发表论文5篇，授权中国发明专利2件，4名研究生参与该项目研究。