木聚糖酶催化反应机理的研究及耐酸碱突变体的设计

Xylanase is a typical glycosidase to break up the glycosidic band in xylan using the acid/base mechanism and have wide applications in pulp, food, and feed industries. Mutating enzyme to have optimal pH at alkaline or acidic environment has great potential in industries. Understanding the reaction mechanism could provide guideline for rational design of protein engineering. Currently, the protonation states of the key residues, ligands and waters have not been directly observed. Thus, the reaction mechanism of xylanase needs to be further elucidated. We have previously obtained the neutron crystallography structures of ligand-free enzymes under different conditions and studied the protonation states of key residues and water. We aims to further obtain neutron-diffracted structures of protein complexes, including the null enzyme-substrate complex, the enzyme-intermediate analog complex and the enzyme-product complex. Neutron diffracted structures can provide detailed informations of hydrogen atoms, which are difficult to visualize using X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance. The above neutron crystal structures will pave the way to studying the reaction mechanism, which involves interactions among xylanase, ligand and water, particularly their protonation states. Our project will adjust the hydrogen network inside the active site to modify the electrostatic environment, which determines the optimal reaction pH during catalysis. This rational design will help to generate a superenzyme whose optimal reaction pH is in the alkaline or acidic condition and has great potential in industry.

木聚糖酶通过酸碱催化机制来断裂木聚糖中糖苷键，在造纸、食品和饲料等行业都有广泛的应用。研究以木聚糖酶为代表的糖苷酶的催化机理，尤其是质子穿梭在断裂糖苷键中的重要作用，对于调节酶最适反应pH值，获得耐酸或耐碱的超级酶，具有重要的理论指导意义。但是，酶活性中心的质子穿梭很难被直接观测到，使得催化机理的研究难以被验证。项目组前期已获得无配体木聚糖酶的中子衍射晶体结构，并将进一步研究无活性突变酶同底物、酶与中间产物类似物、酶与产物等复合物的中子衍射晶体结构，精确地获得用X-光衍射、核磁共振和冷冻电镜等常规手段难以获得的酶、配体和水分子中质子的动态变化，深入地探讨催化机制；通过木聚糖酶与其它糖苷酶水解机制的比较，分析归纳出糖苷酶水解的普遍规律。在上述研究的基础上，尝试改变酶活性中心复杂的氢键网络及其所决定的静电环境，获得热稳定性高、耐酸碱的工程酶，为提高木聚糖酶在工业上的实用性打下理论基础。