Presidential Young Investigator Award

建议从以下分离基因组DNA文库： 詹氏甲烷球菌古细菌。 生物体 是一种极端的嗜温嗜压生物， 位于深海热液喷口附近， 260 atm的压力。（詹氏甲烷球菌是 大多数嗜热生物的DNA 分离，实验室生长最适温度为92 ℃）。 的 这种非凡微生物的DNA将被克隆 用标准的分子生物学方法 程序. 将筛选基因组文库中的 编码氢化酶的基因。 表达 氢化酶基因，使用一种已经 成功克隆产甲烷细菌 基因，将不仅使调查的 控制极端热稳定性的方法， 嗜压性，但也产生了潜在的 有价值的商业酶。 使用的方法 定点突变和随机突变 对温度有不同反应的酶， 将产生压力。 这一系列密切 相关的突变酶将更容易比较， 一组相关的氢化酶分离自内消旋和 嗜热生物 由于这种酶非常 热稳定，应该可以描绘一些 结构-功能关系， 显著的稳定性。 特别令人感兴趣的是 氢化酶的压力增加（特别是 因为这种基因可能具有生理作用， 深海生物的反应）。 压力调节 将详细研究这种酶。 一些 酶被大的压力激活或灭活 但是，这种氢化酶是第一种 在相对较小的变化下， 压力从7.5到260 atm。 动力学结果表明， 拟议的研究将涉及的结构， 酵素 此外，足够的酶将被纯化， 使早期结晶化的尝试成为可能。

It is proposed to isolate a library of genomic DNA from the archaebacter Methanococcus jannaschii. The organism is an extreme thermo- and barophile, its natural habitat being in the vicinity of a deep-sea hydrothermal vent at a pressure of 260 atm. (Methanococcus jannaschii is the most thermophilic organism from which DNA has been isolated with a laboratory growth optimum of 92oC). The DNA from this extraordinary microorganism will be cloned into Escherichia coli using standard molecular biology procedures. The genomic library will be screened for the gene encoding a hydrogenase enzyme. Expression of the hydrogenase gene, using an approach which has already proved successful in cloning methanogenic bacterial genes, will enable not only the investigation of the processes controlling extreme thermostability and barophilicity, but also the production of a potentially valuable commercial enzyme. Using the approaches of site-directed and random mutagenesis a family of similar enzymes with differing responses to temperature and pressure will be generated. This series of closely related mutant enzymes will be far easier to compare than a set of related hydrogenases isolated from meso- and thermophiles. Since the enzyme is extremely thermostable, it should be possible to delineate some of the structure-function relationships which give rise to marked stability. Of particular interest is the response of the hydrogenase to increased pressure (especially since there could be a physiological role for such a response in deep sea organisms). The pressure regulation of this enzyme will be investigated in detail. Some enzymes are activated or inactivated by large pressure changes, but, this hydrogenase is the first enzyme which triples activity over a relatively small change in pressure from 7.5 to 260 atm. The kinetic results of the proposed research will be related to the structure of the enzyme. In addition, enough enzyme will be purified to enable an early attempt at crystallization.