海洋嗜热新菌多结构域褐藻胶裂解酶的结构域功能和催化机理研究

项目名称： 海洋嗜热新菌多结构域褐藻胶裂解酶的结构域功能和催化机理研究

项目编号： No.41506155

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 天文学、地球科学

项目作者： 冀世奇

作者单位： 中国科学院青岛生物能源与过程研究所

项目金额： 23万元

中文摘要： 褐藻胶裂解酶多来自常温菌，在寡糖制备和生物质降解等方面有重要应用价值。AlgAT1是申请人从海洋嗜热新菌Defluviitalea sp. Alg1发酵液中纯化得到的褐藻胶酶，研究表明其N-端含2个预测催化结构域、C-端含5个预测底物结合结构域、60℃酶活半衰期超过40 h、具有对polyM和polyG的双功能酶活性等，多数酶学特征显著不同于已报道褐藻胶酶。因此该酶特殊性质和结构的解析对进一步认识褐藻胶酶的催化机理和热稳定机制意义重大。本课题以AlgAT1为研究对象，拟通过多结构域模块的重组表达，N-端结构域的同源比对、蛋白结构解析和催化活性中心预测，以及定点突变等研究手段，揭示多结构域的功能和催化域的作用机制。本课题的研究将丰富裂解酶的种类，从分子水平上揭示多结构域褐藻胶酶的耐热和催化机制，为褐藻胶酶的分子改造和应用提供理论基础。

中文关键词： 褐藻胶裂解酶；海洋嗜热菌；多结构域；催化机理；热稳定性

英文摘要： Most of alginate lyases were produced from mesophilic bacteria, and they have important applications in alginate-derived oligosaccharide production and biomass degradation. AlgAT1 was one of the alginate lyases isolated from the fermentation broth of an novel thermophilic bacterial strain named Defluviitalea sp. Alg1. This enzyme has two predicted N-catalytic conversed domains and five predicted C-substrate binding conversed domains. It also has an enzyme activity half-life of 40 h, and bifunctional activity towards polyM and polyG. The catalytic activity under high temperature and multi-domain properties were never obvered in other reported alginases. Thus, the special enzymic and structure features are of importance to illustrate the catalytic mechanism and thermostability of alginate lyases. In this project, through expression of the conversed domains indivadully and assembly, multiple sequences alignment, three-dimensional structures determination, catalytic center site prediction, and site-specific mutagenesis study, reveal functions of the multi-domains and the possible catalytic mechanism of AlgAT1. Our research would enrich the categories of alginate lyase, help us to understand the catalytic mechanism and high temperature resistant mechanism in multi-domain enzymes, and supply theoretical basis for the molecular modification and industrial application of alginate lyase.

英文关键词： alginate lyase;marine thermophiles;multi-domain; catalytic mechanism;thermostability