Development of new hypertermophiles and utilization of their products

最近从热液喷口（> ~ 200oc）分离和描述的新微生物具有生态、生理和生物技术方面的兴趣，特别是在超嗜热菌方面。由于在高温高压下栽培困难，这些超高温作物尚未得到利用。在本研究中，我们首先建立了可用于生物技术的80 ~ 100‰的海洋超嗜热菌的分离和培养体系，然后尝试将热稳定酶用于生物技术目的。研究结果如下：(1)采用耐热高压玻璃瓶和丁基橡胶塞，在0.2MPa下，80%H_2和20%CO_2混合气体，研制厌氧培养系统。为了在高达95℃的温度下培养好氧生物，研制了一种由耐热润滑脂和超绝缘材料组成的新型旋转式振动筛。(2)从长崎立花湾浅层热液喷口分离到新的厌氧嗜热细菌热球菌TS100、甲烷球菌TS300、好氧海洋原热菌OKD7和从鹿鹿岛Kodakara岛分离到新的海洋嗜热古细菌Aeropyrus pernix Kl。TS100、TS300、okd7和Kl的最佳温度分别为103、88、80和90-95℃。16SrDNA序列表明，a. pernix Kl属于原始Crenarchaeta， P.maritima OKD7属于原始细菌。(3)从产甲烷菌TS300中纯化并鉴定了非变构CO_2固定酶（磷酸烯醇丙酮酸羧化酶），为心功能不全的诊断提供了依据。a .pernix Kl产生一种有用的细胞外金属蛋白酶，其最佳温度为110ºC，热稳定性极佳（半衰期为120ºC，150min）。热球菌TS100的DNA聚合酶具有很强的耐热性，可用于PCR方法。

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.