Characterization of the Genetic Diversity of Cold-Active Enzymes from Psychrophilic Microorganisms

在自然界中观察到的微生物多样性中，有90%以上没有得到研究或利用，因为这些微生物不能被分离或维持。具有特殊价值的是对嗜冷微生物及其冷活性酶的研究，这些研究由于强调在较高温度下生长的菌株而被忽视了。由于地球和海洋的温度都很低，所以存在着各种各样生理上不同的嗜冷动物。这些生物可以制造冷活性酶，用于低温化学转化的生物技术，用于低温食品加工以防止变质，用于寒冷气候下的生物修复，用于冷水清洗机等。该项目是在无法分离和维持所有现有菌株的情况下获得遗传多样性和冷活性酶的新方法。目的是建立发现和研究从未分离过的新型冷活性微生物编码基因的基本方案。具体目标是开发直接从具有重组DNA实验所需的大小和纯度的微生物或冷环境样品组合中提取DNA的方法。这些技术将通过将基因克隆到表达载体中并检测在新宿主中产生模式酶活性的转化子来验证。这些方法的发展将有助于研究人员发现、研究和使用生理上多样化的微生物的新基因。微生物提供了许多挽救生命的抗生素和有用的酶。不幸的是，这些微生物产品尚未得到充分利用，因为这些微生物从未被分离出来。这个项目将有助于利用微生物携带的遗传潜力。既然分离所有微生物的理想是不可能的，那么另一种选择是从环境材料中提取DNA，直接获取遗传多样性。总DNA的提取将产生以前从未见过的基因，因为它将收集社区中所有微生物的DNA。DNA可以被保存为微型的诺亚方舟，保存着嗜冷基因的多样性，为克隆未来感兴趣的新基因做好准备。从土壤、水和其他样品中提取DNA的方法的发展将使研究人员能够接触到编码工业上有用的酶的基因，并将提供具有不同寻常特征的蛋白质，以便与其他蛋白质结构进行比较。＊＊＊

Brenchley 9513236 More than ninety percent of the Microbial Diversity observed in nature goes unstudied or used because the organisms can not be isolated or maintained. Of special value is the study of psychrophilic microorganisms and their cold-active enzymes that have been overlooked by the emphasis on strains growing at higher temperatures. A wide assortment of physiologically diverse psychrophiles exists because cold temperatures dominate the earth and its oceans. These organisms may make cold-active enzymes with uses in biotechnology for low-temperature chemical conversion, for food processing at low temperature to prevent spoilage, for bioremediation in cold climates, for use in cold-water cleaners, etc. This project is a new approach for gaining access to genetic diversity and cold-active enzymes while not being able to isolate and maintain all existing strains. The objective is to establish the basic protocols for discovering and studying genes encoding novel cold-active enzymes existing microorganisms that have never been isolated. The specific aim is to develop methods for extracting DNA directly from combinations of microorganisms or cold-environment samples that has the size and purity required for recombinant DNA experiments. The techniques will be tested by cloning genes into expression vectors and detecting transformants producing model enzyme activities in a new host. The development of these methods will help investigators discover, study and use new genes from physiologically diverse microorganisms. Microorganisms have provided many life-saving antibiotics and useful enzymes. Unfortunately, full use of these microbial products has gone untapped because the organisms have never been isolated. This project will help make use of the genetic potential carried in microorganisms. Since the ideal of isolating all microorganisms is not possible, then an alternative is to seize the genetic diversity directly by extracting the DNA from environmental materials. The extracti on of total DNA would yield genes never seen before because it would collect DNA from all microorganisms in the community. The DNA could be preserved as miniature Noah's Arks of psychrophilic genetic diversity ready for cloning of new genes of future interest. The development of methods for extracting DNA from soil, water, and other samples would give researchers access to genes encoding industrially useful enzymes and would provide proteins with unusual features for comparison with other protein structures. ***