碳水化合物结合模块(CBM)是广泛存在于糖苷水解酶中的一类重要非催化结构域，目前CBM共分81个家族，其中CBM68由本实验室从耐热普鲁兰酶PulA的晶体结构中首次解析并命名，截去N端CBM68结构域可导致该酶的比活力和最适温度明显下降，但其对普鲁兰酶催化性能的影响机制及底物结合关键氨基酸位点仍待进一步揭示。本项目通过定点突变和融合重组的实验方法，应用ITC、biacore和CD-stopped flow检测手段，从热力学和动力学角度阐明CBM68的底物结合规律，并确定其关键氨基酸位点；同时，结合分子模拟软件（Discovery Studio™）和X-ray晶体结构解析的蛋白质结构分析手段，深入解析各突变酶的底物结合及酶学性质差异，全面揭示CBM68对普鲁兰酶催化性能的影响机制。为CBM结构特性及作用机制的深入研究奠定理论基础，并为普鲁兰酶及相关糖苷水解酶的理性分子改造提供新的理论依据。

Carbohydrate-binding modules (CBM) are important non-catalytic domains widely existed in glycosyl hydrolases. So far, a total of 81 families of CBM have been included in CAZy database. Previously, we have obtained the X-ray crystal structure of a thermostable pullulanase (PulA). The N-terminal domain of its crystal structure was identified as a new type of carbohydrate-binding module and classified to the CBM68 family for the first time. The key amino acids and structural functions of CBM68 remain to be revealed. Truncating the CBM68 resulted in the reduction of specific activity and stability of PulA in our previous study. It indicates that the CBM68 plays important roles in catalytic activity of PulA. Thus, experimental designs will be performed by site-directed mutagenesis and building of fusion recombinant pullulanases. A series of fusion recombinant pullulanases will be built by superposing or replacing the N-terminal (CBM68 and CBM41) of the thermostable pullulanase (PulA) and the acidic pullulanase (Pulnovo) respectively. The difference of the the mutant enzymes in substrate binding will be determined by ITC, biacore and CD - stopped flow. So, the key amino acids of CBM68 and the mechanisms of substrate binding could be revealed through the view of energetic and kinetics. At the same time, the structure based functional mechanism of CBM68 in pullulanase will be further revealed by the comprehensive analysis of bioinformatics (Discovery Studio, DS) and crystal structure. The results of this study could provide theoretical and molecular basis for rational design in order to obtain novel pullulanases and glycosyl hydrolases with enhanced catalytic efficiency.