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嗜热微生物，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到10株好氧超热古菌Aeropyrum pernix，对其生理特性和内含子多样性进行了研究。从熊本县Tuetate温泉中分离到一种新的超嗜热古细菌（Pyrobaculum oguniense），该菌株在90℃以上的好氧条件下生长最多，但在微氧和厌氧条件下也能生长。从伊竹-波宁弧绥约海山热液区分离到一种新的好氧嗜热菌Marinithermus hydrothermalis （opt. temperature . 70C）和一种新的自养氢菌Persephonella hydrobenophila （opt. temperature . 70C）。(2)仙花16S和23S rDNA内含子携带归巢核酸酶基因，表达并鉴定了其性质。两种归巢酶均表现出较高的最适温度（90℃），并能识别特定的长序列。(3)超嗜热古菌a . pernix K1粗提物中存在限制性内切酶活性（ApeK1）。对识别新分裂位点的ApeK1进行了纯化并对其性质进行了表征。ApeK1分子量为40000。最适温度为90℃。通过氨基酸序列分析，鉴定出功能未知的ORF558基因为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.