单催化域几丁质酶CmChi1多催化活性的分子机制研究

The development of broad-specificity chitinase with high catalytic efficiency and simple conversion process is the key to convert polymer chitin into a commercially valuable monosaccharide N-acetylglucosamine (GlcNAc) by single enzyme catalytic method. However, bottlenecks are still occurred in the process of chitin degradation, including lack of high efficient chitinase and unclear catalytic mechanism of these reported broad-specificity chitinases with a single catalytic domain etc.In our previous research, a novel broad-specificity chitinase Cmchi1 which displays endo-chitinase、exo-chitinase and N-acetyl-glucosaminidase activities with a single catalytic domain was screened. The hydrolysis efficiency and GlcNAc purity of Cmchi1 was higher than of other reported broad-specificity chitinases with a single catalytic domain. In this project, catalytic mechanism of broad-specificity chitinase Cmchi1 will be investigated by molecular simulation，molecular docking, molecular dynamics simulation and protein engineering. The structure and conformation of the active center and key amino acid residues involved in substrate recognition will be deep analyzed. The role and synergistic effect of the enzyme function modules will be revealed by expression and purification of Cmchi1 derivatives with splitting function domains. Using site-directed mutagenesis techniques to analyze the key factors involving correlation model of amino acid sequence,enzymatic structure and the broad-specificity catalytic activities. Multi-substrate recognition and broad-specificity catalytic mechanism of the Cmchi1 will be revealed with the combination of theoretical speculation and biochemical characterization. This study will open the door to the possibility of preparation of GlcNAc by single-enzyme method and provide a new insight and meaningful theoretical guidance for the artificial design of high performance broad-specificity chitinase.

开发催化性能优异的多功能几丁质酶是实现单酶法制备N-乙酰氨基葡萄糖的关键，目前报道的单催化域多功能几丁质酶催化效率低且催化机制不明晰。本课题组挖掘到一种单催化域多功能几丁质酶CmChi1，兼具外切酶、内切酶和N-乙酰氨基葡萄糖苷酶活性，且各功能催化活性匹配性好，水解几丁质制备N-乙酰氨基葡萄糖具有转化效率高、产物纯度高的显著特点。本项目拟对CmChi1单催化域多催化活性的分子机制进行研究，将通过同源建模、分子对接、分子动力学模拟、定点突变等手段，研究该酶不同结构域、不同催化活性间的相互作用及协同效应，构建CmChi1参与底物识别和多催化活性关键氨基酸残基的空间构象及其互作网络关系模型，明确关键氨基酸残基影响活性口袋的关键因素，阐明CmChi1底物识别和多催化活性的分子作用机制，为设计和构建各功能催化活性协调的多功能糖苷水解酶提供新的思路和借鉴，为理解和发展多功能糖苷水解酶奠定理论基础。

几丁质酶CmChi1独特的多功能催化特性与催化机制为研究单催化域多功能几丁质酶这类酶的结构与功能关系提供了性能优异的酶资源和理论研究模型，因此，阐明CmChi1底物识别和多催化活性的分子作用机制，有助于了解该类酶底物选择和催化机理的独特性。本项目通过截短表达和点突变对CmChi1的碳水化合物结合模块ChBD-AB的底物结合特性和机制进行了研究，优化获得结合模块ChBD-AB最佳的吸附和固定化条件，明确了参与底物结合的关键氨基酸残基；利用AlphaFold2蛋白质结构预测、分子对接和分子动力学模拟等手段，构建了CmChi1催化域GH18与几丁寡糖的对接图，模拟分析酶与酶-底物复合体的构象变化规律，分析酶与几丁寡糖分子间的对接状态、能量变化与酶底物口袋构象变化的关系，明确关键氨基酸残基影响活性口袋的空间效应、表面静电势及区域结构的关键因素；最后，基于底物结合特性，对碳水化合物结合模块ChBD-AB进行了应用拓展，探究其在酶的固定化与转化方面的应用。

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