基于多结构域和柔性结构特征的普鲁兰酶识别淀粉分支结构的分子机制与性能调控

Starch is kind of polymeric carbohydrate consisting of amylose and amylopectin, where the content of amylopectin accounts for a greater proportion. Pullulanases catalyze the hydrolysis of a-1,6-glucosidic linkages in amylopectin. Because of its debranching ability, the employment of pullulanase during starch saccharification increases the reaction efficiency and utilization ratio of raw material, and improve the product quality. Thus, pullulanase is of importance in the application fields of food, pharmaceutical, energy, and materials as well. Understanding the basic enzymology knowledge concerning the molecular mechanism and structure-function relationship of pullulanase would be important to regulate the enzyme function and the enzymatic reaction. Structure-based revealing the molecular mechanism of selective recognition and interaction between pullulanase and branched structure in amylopectin and even function regulation of the enzyme would be the key points for apparent enhancement of catalytic efficiency of pullulanase. Concerning the structure features of multi domain and flexibility in pullulanase, this research is to elucidate the molecular mechanism of pullulanase selectively recognizing and binding the branched oligosaccharides and catalyzing the hydrolysis reaction by solving X-ray crystal structures and solution structures involving conformational change, to comparatively analyze the relationship between function and structure among homologous pullulanases by the approach combining bioinformatics and molecular modeling, and to enhance the availability and applicability of the pullulanase for industrial application by redesigning the hot spots and functional modules involved in ligand binding and molecular interactions. This research would provide the foundamental theory and research foundation for highly efficient development of pullulanase and even high-value utilization of starch raw materials.

淀粉原料中支链淀粉含量占有较大比例，普鲁兰酶因其水解支链淀粉α-1,6-糖苷键的特性，能够显著提高反应效率和原料利用率并改善产品品质，在食品、制药、能源、材料等领域具有重要的应用价值，认识其催化机制和构效关系的共性酶学分子基础，对于其反应和性能调控具有重要意义。如何基于结构认知探究普鲁兰酶与淀粉分支结构的选择性识别机制，并实现其功能调控，是表征和强化普鲁兰酶催化效率的关键科学问题。本项目拟针对普鲁兰酶的多结构域和无序柔性结构特征，利用静态和动态相结合的蛋白结构分析方法，解析普鲁兰酶选择性识别淀粉分支结构及其催化分支点水解的分子机制；采用生物信息学和分子模拟相结合的序列结构功能系统分析方法，比较普鲁兰酶的性能表征和结构组成规律；基于结构功能关系，对分子设计热点和功能模块进行定向进化和优化组装，拓展和强化其应用性能，为实现普鲁兰酶的高效开发及淀粉糖质资源的高效、高值化利用提供理论和实践基础。

淀粉原料中支链淀粉含量占有较大比例，普鲁兰酶因其水解支链淀粉α-1,6-糖苷键的特性，能够显著提高反应效率和原料利用率并改善产品品质，在食品、制药、能源、材料等领域具有重要的应用价值，认识其催化机制和构效关系的共性酶学分子基础，对于其反应和性能调控具有重要意义。本课题研究提出了一种称为“进化偶联饱和突变”的酶定向进化新策略，基于进化偶联饱和突变策略的普鲁兰酶全序列分析，进一步进行普鲁兰酶结构域氨基酸序列进化偶联分析及改造，获得了具有远端效应并对酶催化活性具有明显影响的新型功能位点；建立了针对酶蛋白稳定性设计提升的组合计算策略、硫醚键预测重构策略等，通过相关策略和方法，设计构建了普鲁兰酶突变体，实现了该酶热稳定性的显著提升，发现了与该酶活性和稳定性调控相关的分子机制；基于前期组合预测算法普鲁兰酶稳定性突变和非天然共价硫醚键重构等研究结果，通过热稳定性预测算法组合预测以及硫醚键“装订”策略，构建了对酶活性有积极影响的正向稳定突变，获得了一系列提高普鲁兰酶BtPul热稳定性的组合突变体；以优势突变体G692M作为研究对象，探究了修饰剂对G692M活性和热稳定性的影响；基于全局调控因子强化策略，提高了重组普鲁兰酶的异源表达水平，并通过系统优化表达条件和调控发酵产酶过程，提高了重组普鲁兰酶的表达产酶活性，进一步通过过表达分子伴侣及融合信号肽策略，促进了突变酶G692M在大肠杆菌系统的分泌效率。相关研究成果发表论文21篇，其中SCI论文20篇；申请中国发明专利3项，授权中国发明专利3项；获得中国石油和化学工业联合会青年科技突出贡献奖1项。研究结果为具有工业属性的普鲁兰酶的分子改造和表达调控等共性技术体系的建立提供理论和实践基础。

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