基于刺参肠道宏基因组文库的新型高效褐藻胶降解酶的筛选及分子机制研究

It has been considered that the production of biobased chemicals and oligosaccharides with biological activity from brown algae has broad application prospects, which is important to solve the world energy crisis and to develop marine drugs industry. However, it is difficult for industrial strains to degrade alginates so that the process could not be applied on a commercial scale. The research of alginate lyases, the key enzymes in the biotransformation of alginate, are relatively delayed, Most of which are endolytic enzymes from cultured microorganisms. There is a lack of excision enzymes with broad substrate specificity. Besides, the research on alginate lyases from uncultured microorganisms is almost in blank state. The project aims to obtain novel high efficient alginate lyases from uncultured microorganisms in the intestin of sea cucumber (Apostichopus japonicus), which is a kind of low temperature animal eating brown macroalgae. In the project, we will also screen and isolate genes coding for novel alginate lyases, and study their catalytic properties after heterologous expression. And then, the structure and function of novel alginate lyases will be studied, which have a broad substrate specificity or cold-adapted characteristics. At last, substrate recognition of alginate lyases with broad substrate specificity and cold-adapted mechanism of cold-adapted alginate lyases will be elucidated by bioinformatics analysis and rational site-specific mutagenesis. It is expected that these results not only lay the microbiological and biotechnological foundation for achieving biotransformation of brown macroalgae for industrial application, but also provide new insights into the theory of alginate degradation.

海藻生物质转化生产生物基化学品和活性寡糖具有广阔的应用前景,对于解决世界性化石能源危机和发展海洋药物产业具有重要意义。但现有工业发酵菌株难以对褐藻胶降解利用，是限制其商业化应用的主要因素。褐藻和褐藻胶生物转化关键酶——褐藻胶裂解酶的研究相对滞后，多为采用传统分离和基于序列同源性筛选可培养微生物中的内切褐藻胶裂解酶，关于海洋未培养微生物中褐藻胶裂解酶的研究几乎空白。本项目拟构建食藻低温动物刺参肠道宏基因组文库，筛选新型高效的褐藻胶裂解酶，同时获得活性表达，研究所筛酶的催化特性；发掘新的具有广泛底物特异性的外切型或适冷褐藻胶裂解酶，解析结构；通过生物信息学分析和理性定点突变，阐明具有广泛底物特异性的外切褐藻胶裂解酶的底物识别机制以及适冷褐藻胶裂解酶的适冷机制。该研究不仅为褐藻生物质转化的工业化应用奠定生物学基础与生物技术基础，而且为微生物降解褐藻胶理论提供新观点。

褐藻发酵生产燃料乙醇，对于解决世界性化石能源危机、粮食问题和环境污染等问题具有重要意义。而且，通过酶降解技术将褐藻胶高值化开发为活性寡糖，具有良好的临床应用前景。目前褐藻和褐藻胶生物转化关键酶——褐藻胶裂解酶的研究相对滞后，缺乏双功能、外切型和适冷等其他催化特性的褐藻胶酶。本项目构建食藻海洋动物建刺参肠道宏基因组文库，结合可培养褐藻胶降解菌基因组研究，筛选（包括未培养）全部微生物的29个褐藻胶裂解酶基因（其中5个基因来自未培养微生物），获得8个新褐藻胶裂解酶，其中3个高效双功能褐藻胶裂解酶：1个新型高效双功能适冷外切褐藻胶裂解rAlys1和2个适冷型内切褐藻胶酶rAlg10和rAlg2。高效双功能适冷外切褐藻胶裂解rAlys1不仅可直接将褐藻胶降解为单糖，而且作为寡糖裂解酶可以降解3糖以上寡糖为单糖，利于工程菌吸收和利用，具有广阔的应用前景。通过同源模建和突变技术，发现双功能适冷外切褐藻胶裂解酶rAlgs1的底物识别与催化中心附近的QIH等保守序列无关，而是与催化中心周围和覆盖于催化中心Loop环非保守序列中带电荷氨基酸密切相关，其适冷特性与Loop环转角处氨基酸有关，这为进一步阐明双功能外切褐藻胶裂解酶的底物识别和适冷机制提供重要参考。该项目筛选出具有适冷特性的高效外切褐藻胶裂解酶和高效双功能内切褐藻胶裂解酶为褐藻生物质转化的工业化应用提供优良酶制剂，而且关于其底物识别和适冷机制研究丰富了肠道微生物降解褐藻胶理论。