木聚糖与木聚糖降解酶相互作用机制及其高效酶水解机理研究

The efficient degradation of the lignocellulose is one of the critical steps in the biochemical conversion of lignocellulose. Xylan interlocks cellulose through hydrogen bonds and chemical cross-links lignin, forming a natural recalcitrance. The efficient enzymatic hydrolysis of xylan has become the focus and nodus of current research because of the complex structure of xylan as well as the diversity of xylan-degrading enzymes. Xylan with different molecular structure and supramolecular structure was prepared by the separation and pretreatment technology of lignocellulose. Moreover, the recombinant xylan-degrading enzymes were prepared by genetic engineering technology. The effects of molecular structure and supramolecular structure of xylan on the catalytic activity and synergistic action of xylan-degrading enzymes were studied, which would clarify the required type and quantity of xylan-degrading enzymes and reveal the interaction mechanism between xylan and xylan-degrading enzymes. Furthermore, based on the structure characteristic of xylan, the composition and proportion of xylan-degrading enzymes was changed to hydrolyze xylan with different structure. The composition and formation kinetics of hydrolysates of xylan were analyzed to clarify the space obstacle and the rate limiting step of enzymatic hydrolysis of xylan with different structure, thus the mechanism of efficient enzymatic hydrolysis of xylan would be revealed. The project can provide theoretical guidance for the construction of efficient lignocellulose degrading enzymes as well as methods for the high value utilization of xylans, thus it has important theoretical significance and application value.

木质纤维素的高效降解是生物化学转化法的关键步骤。木聚糖与纤维素通过氢键紧密结合，而与木质素通过化学键交联，形成了木质纤维素抗降解的天然屏障。由于木聚糖结构的复杂性和木聚糖降解酶种类的多样性，木聚糖的高效酶水解是目前研究的热点和难点。本项目拟通过木质纤维素组分分离和预处理技术制备具有不同分子结构和超分子结构的木聚糖底物，采用基因工程技术制备重组木聚糖降解酶，研究木聚糖分子结构和超分子结构对木聚糖降解酶催化活性和协同作用的影响，阐明木聚糖高效降解所需要酶的种类和数量，揭示木聚糖与木聚糖降解酶相互作用机制。拟基于木聚糖结构特征，调控木聚糖降解酶酶系的组成与比例，通过分析不同结构木聚糖酶水解产物的组成与生成动力学，阐明不同结构木聚糖酶水解过程的空间障碍和限速步骤，揭示木聚糖高效酶水解机理。本项目能为木质纤维素高效降解酶的构建以及木聚糖的高值化利用提供理论指导和方法，具有重要的理论意义和应用价值。

由于木聚糖结构的复杂性和木聚糖降解酶种类的多样性，木聚糖的高效酶水解是目前研究的热点和难点。本项目采用不同的分离方法和预处理手段从甘蔗渣、小麦秸秆和桦木原料中制备了不同分子结构和超分子结构的木聚糖底物，采用基因工程和发酵技术，实现了乙酰木聚糖酯酶、阿魏酸酯酶等木聚糖降解酶在毕赤酵母中的高效表达；通过木聚糖底物与木聚糖降解酶相互作用的研究，发现不同分子结构的木聚糖中侧链取代基团种类和取代程度显著影响木聚糖降解酶的水解效率和协同作用，侧链基团的存在一方面增加了木聚糖的水溶性，但另一方面也限制了木聚糖降解酶与木聚糖分子的结合，侧链取代程度高的木聚糖底物，木聚糖降解酶之间具有较高的协同程度，针对不同分子结构的木聚糖需要不同种类的木聚糖降解酶以实现其高效降解；不同超分子结构的木聚糖底物中所含有的纤维素和木质素等非木聚糖组分对木聚糖降解酶水解效率有较大影响，纤维素和木质素的存在限制了木聚糖降解酶对木聚糖的水解作用，采用预处理技术改变纤维素超微结构和脱除木质纤维素中的木质素组分，能有效促进超分子结构木聚糖底物的酶水解效率；采用木聚糖降解酶和纤维素酶同步协同水解超分子结构木聚糖底物，能提高葡萄糖和木糖等可发酵糖的得率，而采用木聚糖降解酶和纤维素酶逐步协同水解超分子结构木聚糖底物，不仅获得了高附加值的功能糖，而且还提高了纤维素转化率，此外，富含低聚木糖的酶水解液，经过活性炭吸附、水洗、乙醇-水脱附和减压蒸馏等简单步骤能得到纯度为97.29%的木二糖。本项目的研究成果能为木聚糖高效降解酶的构建以及木聚糖的高值化利用提供理论依据和技术方法。