蛋白质定点突变技术应用于木质素对纤维素酶无效吸附的深层机理研究

The high cost of ethanol from lignocellulose hinders its industrialization. The cost from cellulase takes up about 15%-50% of the total expenses. The key to reduce the cost is to decrease the loading of cellulase. Inhibition and adsorption of cellulase to lignin increase the amount of cellulase to guarantee the amount of glucose. But, up till now, the mechanism of adsorption of cellulase to lignin is not clear. Cellulase consists of three components: catalytic domain (CD), with an active site located deep insidea tunnel that spans the entire domain; a much smaller carbohydrate-binding module (CBM) that serves to anchor the enzyme onto the surface of the substrate; and a flexible, highly glycosylated linker peptide that tethers the two globular domains together.It has been shown that CBM plays a great role in the high efficient recovery of cellulase..The aim of the project is to elucidate the role of CBM , CD and structural characteristic of lignin played in adsorption of cellulase to lignin. First, corn stover was acidic pretreated and lignin was isolated gradingly by organic solvent with different polarity. The characteristic of lignin was determined by FT-IR, UV, 13C-NMR and HSQC. The kinetic curve of cellulase adsorption to different lignin was done by dual wavelength. On the other side, site-directed mutagenesis was used to get the CD and CBM from endoglucanase, cellobiohydrolases and β-gulcosidase. Through the analysis of binding force and the number of binding site between CD, CBM and lignin to elucidate the role CD and CBM played in the adsorption of lignin to cellulase. Lastly, by crystallization of CD and CBM before and after being with lignin and X-ray diffraction determination, the binding site could be got. Based on the research, the mechanism of cellulase adsorption to lignin could be elucidated though the relationship between characteristic changes of cellulase and inhibition, adsorption of cellulase to lignin. The results could also make a contribution to the development of industrialization of the second generation ethanol.

纤维乙醇生产的高成本是限制其工业化最重要的原因。木质素对纤维素酶的无效吸附是影响纤维乙醇成本的关键所在，其机制至今没有研究清楚。一般来说，真菌纤维素酶是由一根肽链相连的催化结构域（CD）和吸附结构域(CBM)组成的。研究表明CBM对纤维素酶高效回收有着十分重要的作用。为了研究纤维素酶中的CD和CBM及木质素结构性质在木质素对纤维素酶无效吸附中扮演的角色，一方面对稀酸预处理后玉米秸秆中的木质素进行分级提取和结构表征，另一方面将里氏木霉分泌的纤维素酶进行纯化和蛋白质定点突变，绘制各级木质素与纯化蛋白及其结构域的吸附曲线，找出对纤维素酶吸附最强的木质素进行重点分析以找出木质素结构与无效吸附的关系，将与木质素结合力最强的纯化蛋白结构域进行结构分析，研究被吸附后纯化蛋白及其结构域在性质的变化，并通过X射线衍射分析木质素与结构域在无效吸附中的结合位点。本研究为降低纤维乙醇生产成本提供理论依据。

纤维乙醇生产的高成本是限制其工业化最重要的原因。木质素对纤维素酶的无效吸附是影响纤维乙醇成本的关键所在，其机制至今没有研究清楚。本研究主要分为两个部分，首先一方面通过使用不同的溶剂分级提取稀酸预处理后玉米秸秆、柳枝稷、构树和天然基因突变杨木中的木质素，分别使用FT-IR，GPC和NMR对各级木质素结构进行表征；另一方面分离纯化里氏木霉发酵粗酶液得到外切酶、内切酶和 β-葡萄糖苷酶，通过木质素对纯化酶吸附的初步实验发现木质素对外切酶的吸附能力较强。使用啷个缪尔吸附曲线得到了各级木质素对外切酶吸附的参数，发现玉米秸秆木质素组分中S型和β-O-4的结构含量越高， 对纤维素酶酶解纤维素的影响越大；柳枝稷木质素中的脂肪族羟基以及酚型结构会促进其对外切酶的无效吸附；构树中木质素S/G越高，对外切酶的吸附能力越强；天然基因突变杨木中的木质素，低分子量和分散度(PDI) 的木质素组分对外切酶的吸附能力较强；酚羟基，缩合性芳香族结构会促进其对外切酶的无效吸附。一般来说，真菌纤维素酶是由一根肽链相连的催化结构域（CD）和吸附结构域(CBM)组成的。为了研究纤维素酶中的CD和CBM及木质素结构性质在木质素对纤维素酶无效吸附中扮演的角色，将里氏木霉分泌的外切酶进行蛋白质定点突变，使其只表达催化结构域，将完整的外切酶和只表达催化结构域的外切酶（CDD）添加到纤维素以及纤维素加玉米秸秆分级木质素的体系中研究探讨吸附结构域在木质素吸附外切酶中扮演的角色。结果显示CBH酶解纤维素时，木质素组分的加入会降低酶解体系中纤维二糖的浓度，且第二级木质素对CBH的抑制作用最明显。而CDD酶解时，木质素组分的加入对纤维二糖的浓度影响较小。以上的研究结果说明吸附结构域会增强木质素对外切酶的无效吸附。.本研究为深入研究纤维乙醇生产过程中木质素对纤维素酶酶解效率的影响以及作用机理打下基础，为后续工艺如生物转化工艺提供理论支持和技术指导，对开发生物转化绿色工艺具有重要意义。