海洋新属新种嗜琼胶卵链菌耐热琼胶酶作用机理研究

Agar is a complex polysaccharide which is composed of agarose and agaropectins and is an element in the cell wall of some red algaes. Agar can be degraded by agarase into agarooligosaccharides and neoagarooligosaccharides, which exhibit many biochemical activities. However, agarases have not been used widely for industrial purposes mainly because of their low thermostability. Catenovulum agarivorans YM01 was isolated from coastal of Qingdao and showed high arar degradation activity and thermostability. During our preliminary studies, YM01 was identified to represent a novel genus and species, and the complete genome had been sequenced. According to the annotation of genome sequence, there are 15 genes coding for agar-degrading enzymes, including both alpha-agarase and beta-agarase. In this subject, all the genes of total 15 agarases will be cloned and expressed in E.coli. Enzymology characters and other properties(including substrate specificity, optimum temperature, the degradation product) will be studied and in order to identify the thermostable agarase. On the basis of above, combined with bioinformatics and molecular biological methods, the catalytic and thermostable mechanisms of these enzymes will be studied. I hope this subject should be helpful both to the rich of glycoside hydrolase family, and to the development of new methods for the preparation of specific size of agar-oligosaccharides, and providing a theoretical basis for the design of new thermostable agarase.

琼胶酶是一种具有广泛用途的多糖降解酶，但由于目前发现的琼胶酶均不具有高温稳定性，使其应用受到了很大限制。嗜琼胶卵链菌是申请人所在实验室分离鉴定的一株具有很强琼脂降解能力的海洋新菌，且分泌的琼胶酶具有高温稳定性。对其进行全基因组测序后，经相关蛋白数据库序列比对发现，该菌含有15个预测的琼胶酶编码基因，同时包括α- 琼胶酶和β- 琼胶酶，目前具有该特点的菌株尚未见报道。本课题拟对该菌分泌的系列琼胶酶进行研究，克隆所有琼胶酶的基因序列，进行异源表达和活性检测（包括底物特异性、温度稳定性、产物寡糖分析等），找到耐高温琼胶酶。在此基础上，结合生物信息学和分子生物学方法，对琼胶酶的构效关系和耐高温琼胶酶的高温稳定机制进行研究。希望通过本课题的研究，丰富糖苷水解酶家族组成，开发用于制备特定大小琼胶寡糖的新方法，并为新型耐高温琼胶酶的设计提供理论基础。

海洋细菌新属新种嗜琼胶卵链菌YM01（Catenivulum agarivorans gen. nov. sp. nov.）是本实验室从青岛近海海泥样品中分离得到的一株具有很强琼胶降解能力的细菌。而且该菌分泌的琼胶酶具有很好的高温稳定性。本项目首先对菌株YM01的基因组进行了分析。其基因组大小为5,025,099 bp，包括4,215 个基因、54 个tRNA。菌株YM01中碳水化合物的运输和代谢相关基因占比1.15%，远高于亲缘关系较近的其余菌株，表明菌株YM01在降解利用淀粉、纤维素、结冷胶、琼胶、果胶和木葡聚糖等大分子碳水化合物方面具有明显的基因优势。YM01中预测得到18个与琼胶降解相关的酶，包括13个β-琼胶酶（YM01-1～YM01-13）、2个α-琼胶酶（YM01-14，YM01-15）和3个α-新琼二糖水解酶，并且含有α-琼胶酶降解通路，表明了YM01具有很强的琼胶降解能力。. 对菌株YM01中的10个琼胶酶基因进行重组表达和酶活验证，并对有活性的琼胶酶进行了酶学性质研究。研究结果确定了3个有活性的琼胶酶，分别为YM01-3、YM01-5和YM01-1。其中琼胶酶YM01-3为β-琼胶酶，大小为46.9KD，属于糖苷水解酶16家族，是一个具有高酶活的耐热琼胶酶，其最适反应温度为60℃，并且在50℃～70℃之间都能保持较高的活力，Km和 Vmax分别为3.78 mg/ml，1.1×10（5）U/mg，酶活比已报道的其余琼胶酶高10倍以上。该酶是一种内切酶，降解琼脂糖的终产物为新琼四糖和新琼六糖。对琼胶酶YM01-5的研究结果表明，该酶为β-琼胶酶，大小为88.3KD，属于糖苷水解酶50家族。最适反应温度为37℃，Km和 Vmax分别为222.2 mg/ml和20.8U/mg。YM01-5是一种外切酶，降解琼脂糖的产物主要为新琼二糖。与此同时，YM01-5也能降解不同聚合度的琼胶寡糖和新琼寡糖，产物均为新琼二糖。耐高温琼胶酶YM01-3由于不需要低温储藏和运输，生产时也不需要冷却设备，因此在工业生产上具有广泛应用前景。另外，由于其耐高温的特性，还可以用于科研上从琼脂糖凝胶中回收DNA。琼胶酶YM01-5降解产物只有新琼二糖一种，产物易于纯化和回收。而且可以降解包括琼寡糖、新琼寡糖、琼脂糖等多种底物，因此可以用于工业生产上制备新琼寡糖。