海洋软体动物β-1,3-葡聚糖酶水解及糖基转移机制研究

Laminarin and its degradation product (oligomeric laminarin) have been demonstrated to possess diverse biological activities, making them promising biomaterials with applications in various areas, such as pharmaceutical and functional food. However, properties of laminarin and oligomeric laminarin vary with their structure. Therefore, study aimed at revealing the correlation between the laminarin/oligomeric laminarin structure and their biological activity is the basis for developing seaweed product with high value. Due to endo-β-1,3-glucanases play an important role in those study, more efforts should be put on investigating the properties, structure and function relationship of endo-β-1,3-glucanases with high activity. Previous study has demonstrated that digestive organs of marine molluscs are rich of endo-β-1,3-glucanases, however, few studies regarding endo-β-1,3-glucanases from herbivorous marine animals have been performed. In the previous study, we have cloned a novel endo-β-1,3-glucanase gene with high RPKM value from scallop. In this study, through the integration of genetic engineering, biochemical and biophysical techniques, we are going to carry out in-depth investigation against hydrolysis and transglycosylation mechanism of endo-β-1,3-glucanase. The endo-β-1,3-glucanase will be over-expressed in vitro to carry out enzyme properties study, product analysis and structure determination. Moreover, in combination with site-directed mutagenesis, the function of key amino acids will be determinated and the underlying hydrolysis and transglycosylation mechanism will be elucidated in molecular level. Therefore, this study will provide important enzyme used for laminarine investigation, which will accelerate the development of seaweed polysaccharide source.

海带多糖及其降解产物具有多种生物学活性，在医药卫生、功能食品等领域具有广阔的应用前景。海带多糖或寡糖的结构与活性之间的关系，是该资源高值化开发利用的关键基础。β-1,3-葡聚糖酶是海带寡糖制备及构效关系研究的关键工具酶，因此，高活性β-1,3-葡聚糖酶的性质、结构与功能值得深入研究。前期研究发现，海洋软体动物消化腺中的β-1,3-葡聚糖酶含量较高，但目前针对海洋动物β-1,3-葡聚糖酶的研究较少。申请人从扇贝中获得了一种高表达的新型β-1,3-葡聚糖酶，本项目计划整合基因工程技术、生物化学、生物物理学技术对β-1,3-葡聚糖酶的水解及糖基转移机制进行研究。通过重组表达β-1,3-葡聚糖酶，开展酶学性质、产物组成及酶的空间结构等研究；同时结合定点突变技术，研究关键氨基酸的作用，阐明该酶的水解及糖基转移机制。该研究为海带多糖研究提供重要工具酶，有助于推动海带多糖资源的高值化开发利用。

海洋环境中富含多种多糖资源，如海带多糖、褐藻胶等。研究发现这些多糖的降解产物，寡糖，具有多种生物学活性，在研发新药、功能性食品等领域具有广阔的应用前景。因此，制备高质量的寡糖是深度开发海洋多糖资源的重要环节，而海洋多糖降解酶则是制备高质量寡糖的关键工具酶。在该项目基金的支持下，围绕海洋寡糖制备工具酶，申请人研究了2类，共4种多糖降解酶，主要开展了以下工作：1）首次对扇贝消化腺来源的β-1，3葡聚糖酶Lcf进行了重组表达，并对其酶学性质进行了表征；同时研究了其水解和转糖基反应的特异性。Lcf的水解产物中葡萄糖含量较高，并且Lcf可以以多种底物作为受体发生转糖基反应，有助于新型寡糖的制备。目前，对Lcf的特异性机制的研究还在进行中。2）发掘了3种新型褐藻胶裂解酶，并利用重组表达蛋白对它们进行了酶学性质及降解特点研究：AlyA含有两个具有催化活性的结构域，对polyM表现出降解偏好性；AlyF属于PL6家族但不依赖Ca2+，它可以特异性降解polyG；AlyB含有CBM32结构域，其无明显底物偏好性；并且AlyF和AlyB的产物均以三糖为主，有助于均一性寡糖制备。3) 首次成功解析了AlyB、AlyF、AlyF与三糖和四糖复合物的空间结构，并进行了底物结合机制研究。明确了CBM32结构域和α-helix linker在AlyB中的作用，并提出了AlyB特异性生成三糖的反应模型。同时，提出了AlyF与长链底物的结合机制，明确了决定AlyF特异性生成三糖的关键作用位点（+1，+2和+3位），并基于该位点的结构特点开展了初步的理性改造。以上研究为AlyB、AlyF进一步的改造和应用奠定了坚实的基础。目前，部分研究成果已发表SCI论文5篇，后续关于β-1,3葡聚糖酶和褐藻胶裂解酶的研究还在开展中。

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