Development of new hypertermophiles and utilization of their products

新型超级嗜热菌的开发及其产品的利用

基本信息

批准号：
05556034
负责人：
ISHIDA Yuzaburo
金额：
$ 3.52万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05556034/
关键词：
Hyperthermophile Heat stable enzyme Hydrothermal vent Marine bacteria Archaea Protease DNAポリメラーゼ深海

项目摘要

Recent isolations and descriptions of new microorganisms from the hydrothermal vents(>200ﾟC)have been of ecological, physiological, and biotechnological interest, especially with respect of hyperthermophile. Its has not made use of these hyperthermohpiles because of difficulty in cultivation under hightemperature and pressure. In this study, we firstly establish the isolation and cultivation system of marine hyperthemophiles growing at 80 to 100ﾟC which are available for biotechnology and then attempt to use heat stable enzymes for biotechnological purpose. The results are as follows ;(1)The anaerobic cultivation system is developed by use of heat and high pressure tolerant glass bottlea and butyl rubber stoppers containing a gas mixture of 80%H_2 and 20%CO_2 at 0.2MPa. For culturing aerobic organisms at temperature up to 95ﾟC the new gyrotary shaker is developed by composing of heat torelant grease and super insulating materials.(2)We isolated new anaerobic hypethermophiles Pyrococcus TS100, Methanococcus TS300, aerobic Protothermus maritima OKD7 from shallow hydrothermal vents in Tachibana bay, Nagasaki and the novel marine hyperthermophilic Archaon Aeropyrus pernix Kl from Kodakara island, Kagoshima. Optimum temperature of TS100, TS300, OKD7and Kl are 103,88,80and90-95ﾟC,respectively. The sequences of 16SrDNA shows that A.pernix Kl in a primitive Crenarchaeta and P.maritima OKD7 is a primive the Bacteria.(3)Nonallosteric CO_2 fixing enzyme (phosphoenolpyruvate carboxylase) was purified and characterized from methanogenic bacterium TS300, therefore it might be useful for diagnosis of cardiac insufficiency. A.pernix Kl produces a useful extracellular metalloproteinase which shows optimum temperature of 110ﾟC and theextreme themostability (half life at 120ﾟC,150min). DNA polymerase of Pyrococcus TS100 is a very thermostable and useful for PCR method.

最近从热液喷口（>200°C）中分离和描述的新微生物引起了生态、生理和生物技术方面的兴趣，特别是在超嗜热微生物方面，由于在高温和高温下难以培养，因此尚未使用这些超嗜热微生物。在本研究中，我们首先建立了可用于生物技术的在80至100℃下生长的海洋嗜热菌的分离和培养系统。并尝试将热稳定性酶用于生物技术目的;(1)利用耐热高压玻璃瓶和含有80%H_2和80%H_2的气体混合物的丁基橡胶塞开发了厌氧培养系统。 20%CO_2，0.2MPa，用于在高达 95°C 的温度下培养需氧生物，新型旋转摇床是通过热合成而开发的。 (2)从长崎橘湾浅层热液喷口分离出新型厌氧嗜热菌Pyrococcus TS100、Methanococcus TS300、好氧Protothermus maritima OKD7和鹿儿岛小宝岛新型海洋嗜热菌Archaon Aeropyrus pernix Kl。的温度TS100、TS300、OKD7和Kl分别为103、88、80和90-95°C。16SrDNA序列表明A.pernix Kl在原始Crenarchaeta和P.maritima OKD7中是一种原始细菌。(3)非变构CO_2固定。酶（磷酸烯醇丙酮酸羧化酶）被纯化并A.pernix Kl 是产甲烷菌 TS300 的特征，因此它可用于诊断心功能不全。该酶可产生有用的细胞外金属蛋白酶，其最适温度为 110°C，并且具有极高的稳定性（半衰期为 120°C，150 分钟）。 Pyrococcus TS100 的聚合酶是一种非常耐热的酶，可用于 PCR 方法。