Isolation and Properties of Enzymes from the Hyperthermophile Pyrococcus Furiosus

超嗜热火球菌中酶的分离和性质

• 批准号: 9420228
• 负责人: Evangelos Moudrianakis
• 金额: $ 24万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-15 至 1998-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9420228&HistoricalAwards=false
• 关键词: Isolation; Properties; Enzymes; Hyperthermophile; Pyrococcus

The primary structures of proteins contain the chemical characteristics that determine the native folded conformation having the global free energy minimum for the system in a given microenvironment. Analysis of the pathway(s) of polypeptide chain folding and the nature of the intramolecular interactions that stabilize the final native format are active areas of research in many laboratories. Modern methodologies and technical advances in more classical approaches are now offering a real chance for solving these problems with a high degree of precision. From the standpoint of biological system, the synthesis and thermal stability of enzymes of the hyperthermophilic bacteria and Archaea raise further questi ons about the nature of forces involved in intramolecular stabilization. The experimental system focuses on proteins derived from the hyperthermophile Pyrococcus funosus, an obligate anaerobe isolated from geothermally heated marine sediments off the island of Vulcano, Italy. These organisms grow optimally at 100 C and their enzymes are also maximally active at this temperature. Alpha amylase, a serine protease, a DNAase, and a `dithioglycolase` are the subjects of this study. Work with these proteins will involve their purification from natural and from genetically engineered sources. The purified proteins will be analyzed by enzymologic, spectroscopic, and thermodynamic procedures. Crystallization of some of these enzymes will be attempted. From these studies, a data base will be developed that would allow correlations between structural and catalytic properties characteristic of these thermoactive enzyme systems. This information will be analyzed within the framework of the issue of protein folding and stability. Moreover, in the long run, such understanding may aid in the production of stable enzymes with economic and environmental applications. %%% The primary target of this research is to better our understanding of the process of protein folding. Since the biological specificity and activity of all proteins depends on a precisely folded 3D architecture of the molecule, understanding the rules that govern this process is of fundamental importance both to basic science as well as to biotechnology and medicine. A number of years ago Dr. Anfinsen proposed that the amino acid sequence within a protein chain contained all the thermodynamic information necessary to dictate the final folded state of that protein. Much remains to be understood about the rules governing the mechanics specificity and kinetics of the protein folding pathway(s). The relatively recent introduction of the experimental system of Archaebacteria offers new experimental material for the study of the problem of protein folding and stability. Enzymes from these thermophilic bacteria are stable at high temperatures (up to 100 C); most of the times they are actually inactive at conventional temperatures (15 37 C) and develop their activity only as temperature is raised. The P.I. plans to purify certain enzymes (an amylase, a protease and a DNAase) from such thermophilic archaebacteria and study their catalytic, structural and thermodynamic properties. If successful, these studies should further our understanding of the rules of protein folding and enzyme stability, and might, at some day, contribute towards the engineering of enzymes with improved properties.

蛋白质的一级结构包含的化学特性，决定了天然的折叠构象具有全局自由能最小的系统在一个给定的微环境。分析多肽链折叠的途径和稳定最终天然形式的分子内相互作用的性质是许多实验室的活跃研究领域。现代方法学和技术进步，在更经典的方法，现在提供了一个真实的机会，解决这些问题的高度精度。从生物系统的观点来看，超嗜热菌和嗜热菌的酶的合成和热稳定性进一步引起了对分子内稳定化所涉及的力的性质的疑问。该实验系统的重点是来自超嗜热菌Pyrococcus funosus的蛋白质，Pyrococcus funosus是一种从意大利Vulcano岛外地热加热的海洋沉积物中分离出来的专性厌氧菌。这些生物在100 ℃下生长最佳，它们的酶在此温度下也最活跃。α淀粉酶、丝氨酸蛋白酶、DNA酶和二硫代乙醇酶是本研究的对象。这些蛋白质的工作将涉及它们从天然和遗传工程来源的纯化。纯化的蛋白质将通过酶学、光谱和热力学程序进行分析。将尝试结晶这些酶中的一些。从这些研究中，将开发一个数据库，将允许这些热活性酶系统的结构和催化特性之间的相关性。这些信息将在蛋白质折叠和稳定性问题的框架内进行分析。 此外，从长远来看，这种理解可能有助于生产具有经济和环境应用的稳定酶。 %%% 这项研究的主要目标是更好地了解蛋白质折叠的过程。 由于所有蛋白质的生物特异性和活性都依赖于分子的精确折叠的3D结构，因此了解控制这一过程的规则对于基础科学以及生物技术和医学都具有根本的重要性。 几年前，Anfinsen博士提出，蛋白质链中的氨基酸序列包含了决定该蛋白质最终折叠状态所需的所有热力学信息。 关于蛋白质折叠途径的力学、特异性和动力学的规则仍有许多有待理解的地方。古细菌实验系统的引入为蛋白质折叠和稳定性问题的研究提供了新的实验材料。 这些嗜热菌的酶在高温（高达100 ℃）下是稳定的;大多数时候，它们在常规温度下实际上是无活性的（15 37 C）只有当温度升高时才能发挥它们的活性。 私家侦探计划从这种嗜热古细菌中纯化某些酶（淀粉酶、蛋白酶和DNA酶），并研究它们的催化， 结构和热力学性质。 如果成功的话，这些研究将进一步加深我们对蛋白质折叠和酶稳定性规则的理解，并可能在某一天有助于改进酶的工程特性。