基于嗜热、嗜温及嗜冷菌来源的三种高度同源蛋白酶的温度适应性分子机制研究

项目名称： 基于嗜热、嗜温及嗜冷菌来源的三种高度同源蛋白酶的温度适应性分子机制研究

项目编号： No.31270099

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 唐兵

作者单位： 武汉大学

项目金额： 85万元

中文摘要： 嗜热、嗜温及嗜冷菌来源的酶分别在高温、中温及低温条件下最大限度地发挥其催化功能，以保证微生物在相应温度条件下正常生长。蛋白酶在微生物生理代谢过程中发挥关键作用，对其温度适应分子机制的研究对深入理解微生物的适应机理具有重要的科学意义。本项目利用嗜热、嗜温及嗜冷菌来源的三种蛋白酶（Subtilases）所具备的高同源性这一优势，在对这组酶进行系统比较研究的基础上，通过高效构建涵盖全部非保守区（位点）的系列嵌合体，寻找决定这组酶温度适应差异性的关键区域（位点）并阐明及其作用方式，进而揭示它们的温度适应分子机制，探寻微生物蛋白酶在通过分子进化而实现功能优化的过程中所蕴含的规律，丰富人们对（极端）微生物适应机理的认识。同时，对Subtilases温度适应机制的深入理解，在应用方面将为制备稳定性好且活性高的新型蛋白酶制剂提供新的理论依据。

中文关键词： 嗜热菌；嗜温菌；嗜冷菌；蛋白酶；温度适应

英文摘要： To support normal growth of microorganisms adapted to different temperatures, the enzymes from thermophiles, mesophiles and psychrophiles usually function optimally at high, moderate and low temperatures, respectively. Since proteases play key roles in many physiological processes, the investigation of molecular basis for temperature adaptation of proteases is of great importance for the understanding of the adaptation mechanism of microorganisms. In this research proposal, we are going to probe the temperature adaptation mechanism of proteases using three highly homologous psychrophilic, mesophilic, and thermophilic subtilases. Firstly, we will perform comparative analysis of the properties of the three enzymes, in terms of stability, activity, substrate preferance, autoproteolysis-resistance,etc.. Then, a series of chimera will be constructed by exchanging non-conserved fragments of these enzymes, and will be characterized, in order to identify key structural elements involved in temperature adaptation of the enzymes. Finally, we will perform random shuffling of the genes of the three subtilases, and screen thermostable chimeric enzymes with high low-temperature activity. We hope this study could provide new insight into the molecular basis of subtilases adapted to different temperatures. In addition, the elu

英文关键词： thermophile；mesophile；psychrophile；protease；temperature adaptation