海洋新菌株ZC1的琼胶降解酶系统研究

Through accomplishing several research projects about agarase including the National Natural Science Fundation projects, the applicants have much experience of research on agarase properties and their enzymatic mechanisms. Among all the findings, the marine novel strain ZC1 owns the following special characteristics compared to other reported agar-degrading strains: 1) The production of agarases without the induction of agar, indicating the presence of constitutive agarases; 2) the most putative agarase genes in the reported genomes, with the amount of 40; 3) senven agarases belonged to different glucoside hydrolase families were confirmed after all the 40 putative agarase genes were expressed in E.coli, so strain ZC1 owns the most confirmed agarases agarases of strain ZC1 and study their cellular location and time course of expression using RT-PCR and western bloting, and finally illuminate the agarases' roles and the enzyme system in the agar degradation by strain ZC1 combined with the enzymatic properties of those confirmed agarases. At the mean time, the research on the agarase system will also provide references for the prediction of the agarase systems of other agar-degrading strains based on the comparisons of functional domians composition and regulation sequences of agarase genes. This project will promote the knowledge about the utilization of marine polysaccharides.

通过主持完成多项国家自然科学基金等项目，申请者课题组开展了琼胶酶的性质、作用机制研究；并发现海洋新细菌ZC1不同于已报道的琼胶降解菌株：1）无琼胶诱导下产生高活性琼胶酶，含有组成型琼胶酶；2）其基因组中存在40个推测琼胶酶编码基因，为目前报道具有推测琼胶酶基因数量最多的菌株；3）对所有推测琼胶酶基因进行功能验证，7个推测琼胶酶基因具有琼胶酶解功能，是已报道含有琼胶酶数量最多的菌株。本项目拟进一步通过基因重组表达和免疫检测等技术，研究菌株ZC1中主要琼胶酶的酶学性质、酶解产物和酶反应动力学等，分析主要琼胶酶在琼胶降解中的作用，初步阐释菌株ZC1的琼胶降解酶系统；预期成果将促进对微生物利用海藻多糖机制的认识，促进海藻多糖的开发利用。

菌株ZC1是一株具有多个推测的琼胶酶编码基因的产琼胶酶海洋细菌。本项目主要实现了菌株ZC1中8个推测的琼胶酶编码蛋白的重组表达及其性质研究，发现了E022、E2001、E075和E173等4个编码蛋白属于GH16家族的内切琼胶酶、E575和E576属于GH50家族的外切琼胶酶以及1个二糖降解酶E852。根据酶解琼脂糖的产物，初步推测，在菌株ZC1的琼胶降解酶体系中，4个内切琼胶酶降解琼脂糖主要产生聚合度为4-8的新琼寡糖(中聚合度寡糖)，2个外切琼胶酶再将中聚合度寡糖降解为二糖，后者被二糖酶降解为单糖而被细菌所利用。另外运用蛋白质谱和基因转录分析等方法，初步发现编码蛋白E022、E575、E700和E160可能是菌株ZC1的组成型琼胶酶，E075、E2001和E173应该是琼胶诱导型琼胶酶。本项目初步建立了菌株ZC1的琼胶降解酶体系，丰富了对微生物利用海藻多糖的认识以及海藻多糖降解酶的研究。