新颖顺式环氧琥珀酸水解酶及其催化机制和热稳定性研究

项目名称： 新颖顺式环氧琥珀酸水解酶及其催化机制和热稳定性研究

项目编号： No.31300661

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： 潘海峰

作者单位： 浙江大学

项目金额： 24万元

中文摘要： 顺式环氧琥珀酸水解酶能将顺式环氧琥珀酸转化为酒石酸，产物酒石酸的立体特异性取决于酶学性质。本课题组最近从土壤中分离到一株能转化顺式环氧琥珀酸为L(+)-酒石酸的新菌株，并对该菌的顺式环氧琥珀酸水解酶进行了初步研究。该酶具有比活力高和热稳定性好的优势，但表观分子量、等电点等数据表明，它与现有报道的顺式环氧琥珀酸水解酶有显著差别，是一种新颖的顺式环氧琥珀酸水解酶。本研究拟通过分离纯化、N端测序、基因文库构建和Southern杂交等获得该酶的基因序列，然后通过多序列比对、同源建模、分子对接、定点突变、同位素标记、多肽片段液质联用等揭示其催化机制，最后通过拓扑结构、二级结构、二硫键、氨基酸组成、离子键、氢键、cation-π键、螺旋倾向性和螺旋稳定性探讨其热稳定性机制。本研究不仅对基础酶学具有重要的理论意义，还具有重要的应用价值。

中文关键词： 顺式环氧琥珀酸水解酶；酒石酸；三维结构；催化机制；热稳定性

英文摘要： cis-Epoxysuccinate hydrolase can transform cis-epoxysuccinate to tartaric acid and the stereospecificity of the product is determined by the enzyme. We currently isolate a novel strain from soil which can transform cis-epoxysuccinate to L(+)-tartaric acid and study on its cis-epoxysuccinate hydrolase from primary levels. The enzyme owns higher specific activity and higher temperature stability. Howerver, its molecular weight and pI show significant differences from other reported cis-epoxysuccinate hydrolases, indicating that it is a novel cis-epoxysuccinate hydrolase. This study is to obtain its cDNA sequence through purification of enzyme, N terminal amino acid sequencing, construction of gene library and Southern hybridization，to probe its catalytic mechanism through multiple sequence alignment, homology modelling, molecular docking, site-directed mutagenesis, isotopic labeling experiment and LC/MS analysis of the proteolytic digest, and to propose its thermal stability through topology structure, secondary structure, disulfide bond, amino acid composition, ionic bond, hydrogen bond, cation-π bond, helix propensity and helix stability. This study is not only has theoretical significance for basic enzymology, but also provides application value.

英文关键词： cis-epoxysuccinate hydrolase；tartaric acid；3D structure；catalytic mechanism；thermal stability