面向木质纤维素降解的超宽底物谱多功能糖苷酶催化机制研究

The structure and composition of lignocellulose are extremely complex. To completely hydrolyze this substance into simple sugars, a suite of cellulases and hemicellulases are required to operate in synergy. High cost of enzymes is still the bottleneck of cost-effective enzymatic saccharification of lignocellulosic biomass. Multifunctional glycosidases with more than one kind of catalytic capacities can obviate monofunctional components in lignocellulolytic enzyme cocktail, and work cooperatively to display a higher catalytic efficiency over the.complex of monofunctional enzymes. However, the substrate spectrum of multifunctional glycosidases is still narrow, which can not meet the needs of efficient degradation of lignocellulosic biomass. In our previous research, a novel multifunctional glycosidase BS5 which can hydrolyzed a lot of carbohydrate substrates by the single catalytic domain was screened. The hydrolysis efficiency of enzyme BS5 to alkali pretreated lignocellulose was higher than that of enzyme cocktail. But the catalytic mechanism of this enzyme is not clear. In this.prospect, the function of this enzyme will be investigated through studying on adsorption and degradation performance of different substrates for the enzyme. By molecular simulation and X-ray, the structure, conformation of the active center and key amino acid residues involved in substrate recognition will be analyzed. The role and synergistic effect of the enzyme function domains will be resolved by splitting function domains. Through site-directed mutagenesis techniques, the role of key amino acid will be analyzed. The correlation model of amino acid sequence, enzymatic structure and the multi-substrate catalytic function will be constructed. Multi-substrate recognition and catalytic mechanism of this enzyme will be revealed with the combination of theoretical speculation and experimental data. This research will provide a new insight and meaningful theoretical guidance for the artificial design of high performance multifunctional glycosidase and enrich the basic theory of enzyme multifunction.

木质纤维素结构与组成复杂，其有效降解必须在多种酶类的协同作用下才能完成。多功能糖苷酶能显著减少酶使用种类，提高酶解效率，但目前发现的多功能酶底物谱仍较窄，难以满足木质纤维素高效降解需要。本课题组首次发现一种兼具木质纤维素有效降解必需酶活的多功能糖苷酶BS5，该酶由单催化域呈现多种水解活性，对碱预处理木质纤维素的水解效率高于复合酶系，但该酶的多底物催化机制尚不清晰。本项目以酶BS5为研究对象，表征其功能及水解作用模式；通过分子模拟、X-衍射等手段，分析酶结构特征、活性中心与不同底物结合的微环境变化及关键氨基酸作用；利用酶截短技术及定点突变技术，诠释功能域及其协同效应、关键氨基酸残基在酶多功能性中的作用，建立“序列-结构-多底物催化”的关联模型，阐明酶BS5多功能性的结构基础及催化机制。研究成果将为人工设计高效的多功能糖苷酶提供新的理论依据，对于丰富和发展酶多功能性的基础理论具有重要意义。

木质纤维素结构合组成复杂，其有效降解必须在多种酶类的协同作用下此能完成。多功能糖苷酶能显著减少酶使用成本，提高酶解效率，但目前发现的多功能酶底物谱仍较窄，难以满足木质纤维素高效降解的需要。本项目以自行筛选获得的多功能糖苷酶EG为研究对象，在实现其异源表达的基础上，研究了酶的底物特异性和水解产物特征，该酶对CMC、微晶纤维素、PASC和滤纸等底物均有较好的水解能力，去除底物结合域后，酶EG对微晶纤维素、PASC和滤纸的水解活性提高，而对CMC的水解活性降低；利用X-衍射和同源建模等技术，阐明了酶催化结构域EGP的三维结构特征和活性架构区氨基酸组成，利用丙氨酸扫描，发现活性架构区的芳香族氨基酸影响了酶对底物的亲和性和催化多功能性，阐明了酶多功能性催化的分子机制；利用突变文库构建和迭代饱和突变对该酶进行了分子改造，获得了最佳突变体D70Q/S235W，其外切酶和内切酶活性分别提高了2.1倍和1.7倍，具有更高的热稳定性，而且突变体比原始酶相比，对纤维素的水解效率提高了31.2%，动力学参数测定表明，残基突变提高了酶对底物的亲和力和酶的催化转换系数；分子动力学模拟研究表明，D70Q和S235W的突变改变了酶分子内的相互作用力，增加了酶和底物糖环间的氢键相互作用，由此增加了酶对底物的结合力，提高了酶的催化效率；本研究还利用EVfold等生物信息工具对多功能糖苷酶EGP中共进化残基进行了分析，选定了4对残基进行迭代饱和突变，证明了远端氨基酸也会影响该酶的多功能性，利用分子动力学模拟表明，远端残基的突变改变了酶活性口袋的构型，提高了催化残基与底物分子的接触几率，由此提高了酶的催化活性；利用EDC/IDA修饰可溶可逆的甲基丙烯酸/甲基丙烯酸酯共聚物，实现了多功能糖苷酶的生物亲和固定化，构建了固定化多功能酶溶解催化-沉淀回收的催化体系。本研究共发表论文21篇，其中SCI论文16篇，申请发明专利5项，获授权专利2项，获省部级科研奖2项，1人获得省级人才项目，取得了丰硕的研究成果。