基于宏基因组文库的新壳聚糖酶筛选及计算机辅助合理化设计

项目名称： 基于宏基因组文库的新壳聚糖酶筛选及计算机辅助合理化设计

项目编号： No.31301438

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 程凡升

作者单位： 青岛农业大学

项目金额： 23万元

中文摘要： 本项目以功能食品添加剂壳寡糖的制备难题为出发点，以壳聚糖酶为研究对象，针对其活性、热稳定性低的难题和序列、结构信息少的现实，综合应用极端环境微生物学、宏基因组学、机器学习算法、生物信息学和蛋白质工程展开研究：构建小黄鱼肠道和热环境活性污泥两种特殊生境的宏基因组文库，通过筛选、亚克隆文库构建、异源表达和性质研究，获得典型特征壳聚糖酶；结合已报道的壳聚糖降解酶相关数据，利用基于分子描述符的机器学习算法和结构生物信息学对所获得的功能基因进行分析，研究壳聚糖酶ProSAR和ProSPR形成机制；最后利用（半）理性设计实现壳聚糖酶的蛋白质工程改造，并获得高活力、高热稳定同体的壳聚糖酶。本项目的实施将扩大可应用的新型壳聚糖酶资源；深化壳聚糖酶家族的催化和性质形成机制的认识；本研究所获得热稳定、高活力壳聚糖酶将降低我国虾蟹壳加工的生产成本，对有效利用甲壳素资源和提高海产品的副产物价值有重要意义。

中文关键词： 宏基因组；壳聚糖酶；蛋白质工程；结构模建；生物信息学

英文摘要： The low thermostability and activity of enzymes are the bottlenecks for their applications in industrial scale. Following the wide application of chitooligosaccharide, the urgent need of high thermostable and active chitosanase becomes a block. In addition, the enzyme sources and structural information remain limited and unclear. The present program will utilize extreme environmental biology, metagenomics, bioinformatics and protein engineering methodology comprehensively and cooperatively to study the above issues. Two metagenomic libraries will be constructed targeting to Larimichthys polyactis gut and extreme hot activated sludge microbes; Function-driven screening technology will be applied before and after sub-screening library, and sequencing, heterologously expression, characterization will be used consecutively to evaluate their application potential. Combining with the reported enzymes, the protein sequence activity/propery relationship (ProSA/PR) for the chitosanase catalysis and physicochemical properties will be revealed by machines learning methods and structural bioinformatics. High active and thermostable chitosanase with industrial prospectives will be obtained using protein engineering according to the above guidance. The implementation of this research proposal is meaningful to further our unde

英文关键词： metagenomics；chitosanase；protein engineering；molecular modeling；bioinformatics