κ-卡拉胶酶中碳水化合物结合模块在酶催化中的作用及其底物识别的分子机制

κ-Carrageenase plays a critical role in the production of the κ-carrageenan oligosaccharides, and has extensive application prospects in the pharmaceutical and food industry. It has been generally known that CBM can significantly promote the catalytic efficiencyof the associated enzyme. However, the study of the κ-carrageenan CBM is still scarce. In the previous work, a κ-carrageenan CBM of a κ-carrageenase from Pseudoalteromonas porphyrae LL1 was identified. However, there is little further study about its role in the process of enzyme catalysis and its mechanism of substrate recognition has been not studied. In this study, based on the results of the crystal structure of protein and protein-polysaccharides complexes, a series of structure domains and their substrate binding site mutants will be constructed, expressed and purified, then their enzyme kinetics, polysaccharides binding kinetics and substrate degradation mode will be analyzed. The expected results can comprehensively illuminate the role of the CBM in the enzyme catalysis and its mechanism of substrate binding. This project will elucidate a novel interaction mechanism between proteins and negatively charged sulphated polysaccharides. In the meantime, it will serve as a theoretical underpinning for the rational design of κ-carrageenases with excellent performance, and greatly stimulate the highly efficient development and utilization of κ-carrageenan.

κ-卡拉胶酶是κ-卡拉胶寡糖制备的关键酶，在食品和制药领域有广泛应用前景。碳水化合物结合模块(CBM)可以显著促进相连催化结构域对多糖底物的催化效率，但是至今没有关于κ-卡拉胶CBM的相关研究。本申请人从一株海洋假交替单胞菌的κ-卡拉胶酶中发现并鉴定一个κ-卡拉胶CBM，但该CBM在酶的催化中的作用和机理缺乏深入研究，其与κ-卡拉胶的分子识别机制也未知。所以，本研究拟根据蛋白晶体和蛋白与κ-卡拉胶寡糖复合物晶体结构解析的结果，预测关键底物识别氨基酸，对κ-卡拉胶酶CBM结构域进行突变，分析突变体的酶动力学、底物结合动力学和水解模式，全面揭示并验证该κ-卡拉胶CBM在酶催化中的作用以及与底物特异性识别的分子基础。本项目的预期研究成果不仅提出一个新的带负电荷硫酸多糖与蛋白的相互作用机制，也为理性设计改造高性能κ-卡拉胶酶提供理论基础，进而推动κ-卡拉胶的高效开发利用。

κ-卡拉胶酶可以特异性水解κ-卡拉胶产生寡糖，在食品和制药领域有广泛应用前景。碳水化合物结合模块(CBM)可以显著促进相连催化结构域对多糖底物的催化效率，但是至今没有关于κ-卡拉胶CBM的相关研究。本项目发现海洋假交替单胞菌LL1菌株κ-卡拉胶酶的C末端结构域为新的κ-卡拉胶CBM（PpCBM），并全面揭示了其在酶催化中的作用。本项目结果表明PpCBM可以特异性结合κ-卡拉胶并具有一定的底物破坏作用，而且可以显著提κ-卡拉胶高酶活力和酶热稳定性，并改变酶的底物水解模式。即无论以溶解和胶体状态κ-卡拉胶为底物时，PpCBM都可以使酶的底物水解模式倾向于进行性水解。然后，本项目又初步探究酶催化和底物结合的分子机制。通过同源建模和定点突变分析发现该κ-卡拉胶中的关键催化氨基酸为E162、D164和E167，而PpCBM中的关键κ-卡拉胶结合位点而为E376和K359，其中带正电荷的K359很可能涉及与κ-卡拉胶的硫酸集团的离子相互作用。在本项目的支持下，本人还构建重组型短芽孢杆菌分泌表达系统，获得高酶活力的κ-和ι-卡拉胶酶以并制备相应的卡拉胶寡糖。此外，还进一步拓展CBM的应用研究，通过融合菊粉CBM结构域构建耐热菊粉酶内切酶工程蛋白水解菊粉高效生产益生果寡糖。综上，本项目的研究成果揭示了κ-卡拉胶CBM的作用和其初步的机制，并为卡拉胶寡糖的高效生产提供基础。