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具有生物技术应用价值的新型超嗜热微生物，并开发这些超嗜热基因的超催化功能。(1)从日本长崎立花湾海底热液喷口分离到10株嗜热古菌Aeropyrum pernix，对其生理特性和内含子多样性进行了研究。从日本熊本县Tuetate温泉中分离到一种新的超嗜热古菌Pyrobaculum oguniense，该菌在微氧和厌氧条件下也能生长，但在有氧条件下生长的温度最高可达90 ℃以上。在伊豆小笠原岛弧水予海山的热液区，发现了一种新的好氧嗜热菌Marinithermus hydrothermalis（最适温度为200 ℃）。70 ℃）和一种新的自养产氢细菌Persephonella hydrobenophila（op.temper.70 ℃）。70C）进行分离和表征。(2)A. pernix携带归巢核酸内切酶基因，该基因被表达并表征了性质。两种归巢核酸内切酶均显示出较高的最适温度（90 ℃），并识别特定的长序列。(3)超嗜热古菌A.在粗提物中发现pernix K1 a限制性内切酶活性（ApeK1）。纯化了识别新分裂位点的ApeK1，并对其性质进行了表征。ApeK1的分子量为40，000。最适温度为90 ℃。功能未知的A.经氨基酸序列分析，确定pernixK1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.