海洋细菌来源的PL14家族褐藻胶裂解酶的性质、结构和催化机制研究

Alginate lyases are the main enzymes involved in the degradation of alginate in the ocean, playing an important role in the degradation and recycling of alginate. They are also widely applied in biotechnology and medicine. Alginate lyases are distributed in 7 polysaccharide lyase (PL) families, having diversity in sequence, structure and catalytic mechanism.Till now, The property, structure or catalytic mechanism of the PL14 alginate lyases from marine bacteria have not been reported. This program aims to illustrate the property, structure or catalytic mechanism of the PL14 alginate lyases from marine bacteria. Based on our previous research on the PL14 alginate lyases from marine bacteria, more than 10 genes of PL14 alginate lyases from marine bacteria will be further cloned or synthesized, expressed, purified and characterized. The structures of bacterial PL14 alginate lyase and of the complex of enzyme-substrate will be solved. The catalytic mechanism of marine bacterial PL14 alginate lyase will be illustrated by structural analysis in combine with mutational analysis. The results may lead to the finding of new structure and new catalytic mechanism of alginate lyase. This study will help in revealing the process and mechanism of alginate degradation driven by marine bacteria, and also lay a foundation for the exploration of new alginate lyase resource.

褐藻胶裂解酶是海洋中参与褐藻胶降解的主要酶类，在褐藻胶降解与循环中发挥重要作用，在生物技术和医药领域也有广泛的用途。褐藻胶裂解酶分布在7个多糖裂解酶家族中，其一维序列、三维结构和催化的分子机制都具有多样性。目前，海洋细菌产的PL14家族褐藻胶裂解酶的酶学特性、三维结构和催化机制尚未见报道。本项目旨在阐明海洋细菌来源的PL14家族褐藻胶裂解酶的性质、结构和催化机制。首先在前期对细菌PL14褐藻胶裂解酶研究的基础上，进一步克隆/合成10条以上海洋细菌PL14褐藻胶裂解酶基因，异源表达和分离纯化后，研究并揭示其酶学特性；解析褐藻胶裂解酶以及酶-底物复合物的晶体结构；通过结构分析以及定点突变等阐明海洋细菌PL14家族褐藻胶裂解酶的催化机制。研究结果有望发现褐藻胶裂解酶的新结构和新催化机制，有助于揭示海洋细菌参与并驱动褐藻多糖的降解与循环的过程与机制，也将为新型褐藻胶裂解酶资源的开发与利用奠定基础。

褐藻胶是海洋中重要的有机碳，海洋细菌褐藻胶裂解酶在褐藻胶的降解中发挥着重要的作用。目前PL32、PL34 和PL36家族褐藻胶裂解酶的结构和催化机制尚未阐明。经过4年研究，本项目取得如下研究成果：（1）首次揭示了PL36家族褐藻胶裂解酶的性质、结构和催化机制；（2）揭示了极地来源的适冷褐藻胶裂解酶AlyC3的结构、盐适应机制及其催化过程与机制；（3）揭示了南北极细菌的褐藻胶代谢途径；（4）阐明了一个新型具有双催化结构域的褐藻胶裂解酶的特征及其对降解褐藻胶的协同降解；（5）揭示了一个新型褐藻胶裂解酶在褐藻寡糖制备中的应用潜力；（6）综述分析了假交替单胞菌属细菌对褐藻胶的降解和利用情况。这些研究成果揭示了海洋细菌褐藻胶裂解酶的结构、催化机制、环境适应机制和应用潜力，以及南北极细菌的褐藻胶代谢途径，对阐明海洋细菌在海洋褐藻胶降解和循环中的作用具有重要意义。