耐热普鲁兰酶新底物结合域CBM68对普鲁兰酶催化性能的影响机制

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.

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。

普鲁兰酶可特异水解淀粉中α-1,6糖苷键，有效提高糖化率，具有良好的应用前景。普鲁兰酶N端的底物结合域（CBM）通常对酶的底物结合及酶学性质具有重要影响。本项目针对前期确定的关键丙氨酸突变酶Y14A、V91A、G92A和R96A，综合ITC和SPR技术分别测定了各突变酶的底物结合特性，结果显示 PulA-D413N的KD值分别是V91A-D413N和G92A-D413N的1.58倍和2.19倍，而Y14A-D413N和R96A-D413N的KD值分别是PulA-D413N的1.04倍和1.69倍。对4个位点分别定向突变成不同类型氨基酸，酶学性质变化结果显示91和92位残基在CBM68的作用相对更为重要，并且91和92位分别突变为极性酸性氨基酸E和碱性氨基酸R更有利于普鲁兰酶对底物的结合与催化。应用DS软件对各突变位点进行作用力的计算分析，显示范德华力和氢键是第14位点与麦芽三糖结合的主要作用力，范德华力是改变第91位点与麦芽三糖结合的主要作用力，影响第92位点和96位点对麦芽三糖结合作用力，包括氢键和范德华力的共同作用。同时，本项目以CBM68整体结构域为研究对象，构建12个融合重组酶对比了CBM68与CBM41及CBM41-X45针对普鲁兰酶催化性能产生的不同影响。结果显示，CBM68与CBM41和CBM41-X45相比，对普鲁兰酶的耐热性保持具有更积极影响，同时更利于普鲁兰酶在相对中性的pH条件下反应。而CBM41或CBM41-X45与CBM68同样对普鲁兰酶的底物结合及催化效率具有促进作用，但其相比于CBM68，对耐热性的维持显示了不利影响。另外，不同CBM结构域与不同催化结构域的组合会产生更新、更具潜力的突变体。综上，本项目由点及面的全面解析了新型底物结合域CBM68的主要生物学功能及其对普鲁兰酶催化性能的影响机制，为普鲁兰酶的定向分子改造提供了理论依据。