新型低钙离子依赖碱性果胶酸裂解酶作用机制及其与碱适应性关系研究

项目名称： 新型低钙离子依赖碱性果胶酸裂解酶作用机制及其与碱适应性关系研究

项目编号： No.31300667

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 周成

作者单位： 中国科学院微生物研究所

项目金额： 25万元

中文摘要： 果胶酸裂解酶是许多植物发病机理的主要毒力因子且在许多绿色工业过程中具有重要应用价值。Bacillus sp.N16-5碱性果胶酸裂解酶PelA是首个低Ca2+依赖和具有最高碱适应性的果胶酸裂解酶,目前对于其低Ca2+依赖的底物结合与催化的作用机制及其碱适应分子机制还未有研究，这种低Ca2+依赖特性与其高碱适应性之间的关系也未可知。本课题拟通过对PelA及其与底物和Ca2+复合物结构的解析和比对及突变分析，揭示PelA不同于已报道果胶酸裂解酶的底物结合和催化模式；通过基于结构信息的比对、关键位点突变和pKa值分析及不同pH下Ca2+亲和性分析等，阐明PelA的Ca2+和底物结合域及催化活性中心等适应高碱性的分子和结构特征，揭示低Ca2+依赖性与其高碱适应性的关系。本研究可揭示果胶酸裂解酶新的底物结合与催化作用机制及其碱适应分子机制，并可为果胶酸裂解酶基于工业应用的分子改造提供理论指导。

中文关键词： 碱性果胶酸裂解酶；低钙离子依赖；结构特征；底物结合与催化；碱适应特征

英文摘要： Pectate lyases are believed to be the major virulence factor of pathogenesis in a broad range of plants and have important application in many biological industries. The pectate lyase PelA from Bacillus sp.N16-5 is the most alkaline and the first low Ca2+ dependent pectate lyase reported. Until now, the mechanism of substrate binding and catalysis for this low Ca2+ dependent pectate lyase has not been studied. The alkaline adaptation mechanism of pectate lyase is still unknown. And the relationship between alkaline adaptation and low Ca2+-dependence is also not clear. In this study, firstly we want to reveal the novel mechanism of substrate binding and catalysis of low Ca2+ dependent pectate lyase by crystal structure resolving and sequence and mutagenesis analysis of PelA and its complex with substrate and Ca2+.Secondly, we want to clarify the alkaline adaptation mechanism of pectate lyase by structure comparison, site mutagenesis and pKa analysis. Then combining the Ca2+ affinity in different pH and molecular evolution analysis and the alkaline adaptation mechanism of PelA，we want to reveal the relationship between alkaline adaptation and low Ca2+ dependence of pectate lyase. These researches will reveal a novel mechanism of substrate binding and catalysis of pectate lyase and alkaline adaptation mechanism of

英文关键词： alkaline pectate lyase；low calcium dependence；structure characteristic；substrate binding and catalysis；alkaline adaptation characteristic