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适用于生物技术的新型高温微生物，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中分离到10株好氧高温古细菌Aeropyrum Pernix，对其生理特性和内含子多样性进行了研究。从熊本县图埃塔温泉中分离到一株新的嗜热古生菌--焦杆菌，该菌能在90℃以上的有氧条件下生长，但也能在微氧和厌氧条件下生长。在伊豆-博宁弧水育海山的热液中，发现了一种新的好氧嗜热细菌Marinithermus ahthermal(Opt.临时工。70c)和一种新的自养型氢细菌Persephelella hybenophila(Opt.临时工。(2)Pernix的16S和23S rDNA内含子携带有归巢内切酶基因，并对其进行了表达和性质鉴定。两种同源内切酶均表现出较高的最适温度(90℃)，并能识别特定的长序列。(3)在嗜热古细菌ApeK1的粗提物中发现了一种限制性内切酶活性(ApeK1)。对识别新裂解位点的ApeK1进行了纯化，并对其性质进行了鉴定。ApeK1的相对分子质量为40,000。最适温度为90℃。通过氨基酸序列分析，确定未知功能基因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.