報告書番号

U87053

タイトル（和文）

水素生産嫌気性細菌の育種－セルラーゼ遺伝子のクローニング－

タイトル（英文）

CLONING OF CLOSTRIDIUM CELLOBIOPARUM CELLUASE GENE

概要 （図表や脚注は「報告書全文」に掲載しております）

バイオテクノロジーの電気事業への応用のひとつとして，嫌気発酵による水素生産の研究を行った。ここではセルロースを分解して水素を活発に生産する嫌気性細菌を育種する目的で，水素生産菌と類緑の嫌気性セルロース分解菌Ｃｌｏｓｔｒｉｄｕｍｃｅｌｌｏｂｉｏｐａｒｕｍのセルラーゼ遺伝子をクローニングした。２種類のセルラーゼ遺伝子がクローニングされ，そのうちセルラーゼ活性が強いものについて制限酵素切断点地図を作成した。このセルラーゼ遺伝子の大きさは２７ｋｂ以下であった。この遺伝子によって生産される酵素はカルボキシメチルセルロースおよびｐ－ニトロフェニル－β－Ｄ－セロビオシドのみを分解したことからエンドグルカナーゼと考えられた。

概要 （英文）

RECENT RAPID ADVANCE IN BIOTECHNOLOGY HAS EFFECTED ON VARIOUS INDUSTRIAL FIELDS. SO,IT ALSO MAY BECOME ONE OF IMPORTANT TECHNIQUES FOR ELECTRIC POWER INDUSTRY IN FUTURE. HYDROGEN IS EXPECTED TO BECOME AN IMPORTANT ENERGY BECAUSE OF CLEANLINESS AND APPLICABILITY FOR FUEL CELL. FROM A VIEW POINT OF ECONOMICAL HYDROGEN PRODUCTION,THE MOST PRACTICAL BIOLOGICAL PROCESS MAY BE AN ANAEROBIC FERMENTATION,SINCE SOME ANAEROBIC BACTERIA CAN PRODUCE HYDROGEN FROM CELLULOSIC SUBSTACTE WHICH ARE ONLY RENEWABLE RESOURCES AVAILABLE IN LARGE QUANTITIES. THE RESEARCH HAS BEEN CONDUCTED ON BREEDING ANAEROBIC BACTERIA WHICH HAS AN EMPHASIZED ABILITY ON HYDROGEN PRODUCTION FROM CELLULOSE BY USING GENE MANIPULATION METHODS. IN THE PRESENT WORK,THE CLOSING OF C.CELLOBIOPARUM CELLULASE GENE WILL BE DESCRIBED AS THE FIRST STAGE OF THIS PROJECT. CHROMOSOMAL DNA FROM C.CELLOBIOPARUM WAS PARTIALLY DIGESTED BY RESTRICTION ENDONUCLEASE HINDIII. THEN DNA FRAGMENTS WERE LIGHTED WITH CLONING VECTOR PUC19. E.COIL JM109 WAS TRANSFORMED WITH LIGATED DNA. RECOMBINANTS PRODUCING CELLULASE WERE SELECTED BY THE CONGO RED METHOD. THREE RECOMBINANTS MADE CLEAR ZONES ON CONGO RED PLATES. BUT STUDIES ON RESTRICTION PATTERNS REVEALED THE TWO OF THEM HARBORED THE SAME DNA FRAGMENT. THE PLASMID WITH HIGHLY ACTIVE CELLULASE GENE WAS NAMED PCMC1. THE CELLULASE GENE WAS ENCODED IN 2.7KB. FRAGMENT IN PCMC1. THE CELLULASEENCODED ON PCMC1 DEGRADED CARBOXYMETHYLCELLULOSE AND P-NITROPHENY1-BETA-D-CELLOBIOSIDE. BUT IT WAS INACTIVE FOR AVICEL. THESE RESULTS DEMONSTRATED THAT THE GENE WAS DIRECTING THE SYNTHESIS OF ENDOGLUCANASE.

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