Isolation of novel thermophilic organisms from marine hydrothermal environments and the development of new catalytic functions

本研究的目的是从海洋热液环境中分离出对生物技术有用的新型超高温微生物，并开发这些嗜热基因的超催化功能。(1)从长崎县立花湾海底热液喷口分离出10株需氧超高温古菌Aeropyrum pernix，对其生理特性和内含子多样性进行了表征。从熊本县的Tuetate温泉中分离出一株新的超嗜热古细菌Pyrobaculum ouniense，该细菌在90℃以上的有氧条件下最能生长，尽管该分离株在微氧和厌氧条件下也能生长。从伊豆-小笠原岛弧水阳海山热液场中分离和鉴定了一株新的好氧嗜热细菌Marinithermus water Thermalis (opt. 70C)和一株新的自养产氢细菌Persephonella Hydrobenophila (opt. 70C)。(2) A. pernix的16S和23S rDNA内含子携带归巢核酸内切酶基因,表达并表征了属性。两种归巢核酸内切酶都表现出较高的最适温度(90℃)并识别特定的长序列。(3)在超嗜热古菌A. pernix K1的粗提物中发现了限制性内切酶活性(ApeK1)。纯化识别新分裂位点的 ApeK1 并表征其特性。 ApeK1的分子量为40,000。最适温度为90℃。根据氨基酸序列分析结果，鉴定出A. pernixK1功能未知的ORF558为ApeK1基因。

The purposes of this study are the isolation of useful new hyperthermophilic microorganisms for biotechnology from marine hydrothermal environments and the development of super catalytic functions of these thermophilic genes.(1) Ten strains of aerobic hyperthermophilic archaeon Aeropyrum pernix were isolated from the submarine hydrothermal vents in Tachibana Bay, Nagasaki Prefecture, characterized the physiological properties and intron diversity. A new hyperthermophilic archaeon Pyrobaculum oguniense was isolated from Tuetate hotspring in Kumamoto Prefecture, which could grow most under aerobic condition over 90C although the isolate was also able to grow under microaerobic and anaerobic conditions. From the hydrothermal field at the Suiyo Seamount in the Izu-Bonin Arc, a new aerobic thermophilic bacterium Marinithermus hydrothermalis (opt. temp. 70C) and a new autotrophic hydrogen bacterium Persephonella hydrobenophila (opt. temp. 70C) were isolated and characterized.(2) The 16S and 23S rDNA introns of A. pernix carried homing endonuclease genes which were expressed and characterized the properties. Both homing endonucleases showed high optimum temperature (90C) and recognized the specific long sequences.(3) In hyperthermophilic archaeon A. pernix K1 a restriction enzyme activity (ApeK1) was found in crude extract. ApeK1 recognizing new split site was purified and characterized the properties. Molecular weight of ApeK1 was 40,000. The optimum temperature was 90C. The function unknown ORF558 of A. pernixK1 was identified the gene of ApeK1 from the result of amino acid sequence analysis.