Improving the catalytic activity of a thermostable anthranilate phosphoribosyl transferase by directed evolution

通过定向进化提高热稳定邻氨基苯甲酸磷酸核糖基转移酶的催化活性

• 批准号: 5427583
• 负责人: Professor Dr. Claus Seidel
• 金额: --
• 依托单位: Lehrstuhl für Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5427583?language=en
• 关键词: Improving; catalytic; activity; thermostable; anthranilate

Enzymes from hyperthermophilic microorganisms are extremely thermostable and catalytically highly active at temperatures close to the boiling point of water. However, most hyperthermophilic enzymes are only marginally active at the optimum growth temperatures of mesophiles. It will be attempted to increase the low activity at 37°C of anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Sulfolobus solfataricus (sTrpD) by directed evolution. To this end, cells of an auxotrophic Escherichia coli strain lacking a functional trpD gene will be transformed with a strpD gene library that has been generated by random mutagenesis, plated on minimal medium without tryptophan and incubated at 37°C. sTrpD variants with improved catalytic activities compared to the wild-type enzyme will be identified by a faster growth of the transformed cells. The characterisation of the purified variants by multiparameter fluorescence detection (MFD) of single molecules, steady-state enzyme kinetics, stopped-flow techniques, chemical and thermal unfolding experiments, and limited proteolysis will show to what extent and by what mechanisms catalytic activity and conformational stability are coupled. Moreover, MFD will quantitatively describe the extent and the dynamics of the structural changes during catalysis as well as their relation to enzymatic activity. The obtained results will be analysed on the basis of the known X-ray structure of sTrpD.

来自超嗜热微生物的酶在接近水的沸点的温度下具有极高的热稳定性和催化活性。然而，大多数超嗜热酶在中温菌的最佳生长温度下仅具有少量活性。将尝试通过定向进化来增加来自超嗜热古菌硫磺硫化叶菌（Sulfolobus solfataricus）的邻氨基苯甲酸磷酸核糖基转移酶（sTrpD）在37°C下的低活性。为此，将缺乏功能性trpD基因的营养缺陷型大肠杆菌菌株的细胞用通过随机诱变产生的strpD基因文库转化，接种在不含色氨酸的基本培养基上并在37°C下孵育。与野生型酶相比具有改进的催化活性的sTrpD变体将通过转化细胞的更快生长来鉴定。通过单分子的多参数荧光检测（MFD）、稳态酶动力学、停流技术、化学和热展开实验以及有限的蛋白水解对纯化的变体进行表征，将显示催化活性和构象稳定性在何种程度上以及通过何种机制耦合。此外，MFD将定量描述催化过程中结构变化的程度和动力学以及它们与酶活性的关系。将根据已知的sTrpD的X射线结构对所得结果进行分析。