纤维素酶在木质素上的吸附规律和机制及弱木质素吸附性酶的构建

Now, bioconversion of renewable lignocellulose for producing liquid flues and chemicals is a hot topic in the world, in which, a key step is that lignocellulose is hydrolyzed to fermentable sugar using cellulase. In the enzymatic hydrolysis of lignocellulose, however, the non-productive adsorption of enzymes onto lignin affects the enzymatic hydrolysis of cellulosic substrates. The studies about the adsorption of cellulase onto lignin from literatures were focused on the influence of lignin on free protein concentration in the enzyme solution and different enzymatic activity, and focused on studying the rule and mechanism of adsorption of limited several main components in cellulase such as Cellobiohydrolase(CBH), endoglucanase(EG) and β-glucosidase onto lignin. It is difficult to fully reveal the rule of adsorption of cellulase with a complex system onto lignin and mechanism of the effect of lignin adsorption on enzymatic hydrolysis using the results from literatures. . In this project, adsorption rules of different source cellulase and different components in crude cellulase liquor such as various enzyme components and non-enzymatic proteins onto lignin will be systematically studied at the level of the whole enzymatic system of cellulase rather than several main enzyme components, and and influence of the lignin adsorption on enzymatic activity of different enzyme components will be evaluated. The interactions of different protein during adsorption of enzymes to lignin will also be investigated in the multi protein system. The differences on adsorption of different enzyme components onto lignin and on effect of the adsorption on catalytic ability of different enzyme components will be compared and the differences mechanism will be elucidated from the protein structure and properties of different enzyme angle. Based on the results, the enzyme with the ability of weak-lignin-adsorption and strong-cellulose-adsorption will be constructed by enzymatic engineering modification, and the cellulase with high-efficiency degrading enzymatic system will be built according to the characteristics of different cellulosic substrates for achieving effective degradation of cellulosic substrates under low dosage of cellulase. The study is of great significance for modifying cellulase system by rational design method and more understanding the adsorption mechanism of enzyme onto lignin.

利用木质纤维素生物转化生产液体燃料和化学品是当前国际上研究的热点，其中将纤维底物用酶水解成可发酵糖是关键。已知木质素对酶的非生产性吸附影响纤维素的酶解效率，但已有木质素对酶吸附研究集中在木质素对纤维素酶液中蛋白量和酶活的影响以及对几个主要酶组分的吸附和机制研究，无法全面反映木质素对具有复杂酶系组成的纤维素酶的吸附规律及对纤维素酶解的影响机制。项目拟在全酶系水平上，系统研究木质素对不同来源的纤维素酶及其酶系中不同蛋白组分的吸附规律及对不同酶组分活性的影响机制，揭示多蛋白体系中不同蛋白在木质素吸附中的相互影响规律，从酶的结构和性质角度阐明木质素对不同酶组分吸附和对酶活性影响存在差异性的机制，在此基础上，通过构建“弱木质素吸附性和强纤维素吸附性”的酶组分和针对不同底物的高效降解酶系，实现低酶用量下纤维底物的高效水解。研究对后续通过理性设计改造纤维素酶和纤维素酶系、完善木质素对纤维素酶的吸附机制等具有重要意义。

在木质纤维素生物转化过程中，木质素对酶的非生产性吸附降低了纤维素酶的水解效率。但目前国内外已有研究缺乏木质素对纤维素酶系中不同酶蛋白的吸附差异性及其机制的深入解析，对木质素吸附影响不同酶组分催化活性的机制也尚未阐明。针对上述背景，本项目系统解析了草酸青霉纤维素酶的酶系组成；从全酶系角度研究了木质素对草酸青霉纤维素酶不同酶的吸附特征，并对吸附量较大且对纤维素水解有重要影响的主要酶组分CBH、EG、Xyn（木聚糖酶）等在木质素上的吸附规律、对不同酶催化能力的影响和机制等进行了详细研究，证实木质素对CBHI、EGⅡ和Xyn10A的吸附过程都遵循二级动力学模型，吸附热力学过程都遵循D-R模型，木质素对它们的吸附都属于物理性的吸附；改变反应体系的pH值和温度会影响蛋白在木质素上的吸附能力和解吸附性能。酶蛋白被木质素吸附后形成了较为稳定的“木质素-蛋白”结合体，在酶水解条件下蛋白难以从木质素上脱析出来。CBH Ⅰ与木质素结合后仍保持一定的催化活性，而EG和Xyn10A与木质素结合后对酶活性影响很大；进一步研究证明CBH和EG的CBM区在与木质素结合中发挥着重要作用，分别通过点突变对CBHⅠ和EGⅡ的CBM区的主要结合位点的氨基酸进行理性改造，获得了对木质素亲和力减弱的突变酶，利用突变酶水解木质纤维素可以获得更高糖产量。此外，还对不同来源的BGL和Xyn10A的功能区分别进行了重组，构建出了与木质素结合能力降低的工程酶，例如，将黑曲霉木聚糖酶Xyn11B的CD、烟曲霉Xyn10A的linker以及草酸青霉Xyn10A的CBM进行融合，构建的重组酶与木质素具有较低的结合能力并对预处理后玉米秸秆木聚糖表现出良好降解能力。预处理方法、预处理强度等都影响木质素对酶的吸附能力。项目研究对完善木质素对酶的吸附机制、从理论上指导寻找有效减少木质素吸附的方法、构建更高效纤维素降解酶系等具有重要科学意义。