報告書番号

U88055

タイトル（和文）

水素生産嫌気性細菌の育種－組換え体大腸菌Ｅ．ｃｏｌｉＪＭ１０９におけるセルロース分解酵素の菌体外放出－

タイトル（英文）

CELLULASE SECRETION FROM TRANSFORMED ESCHERICHIA COLI JM109

概要 （図表や脚注は「報告書全文」に掲載しております）

本研究では植物の主要構成成分であるセルロースを分解し，効率よく水素を生産する微生物の遺伝子組換え技術による育種を目指している。ここではこれまでに取得したセルロース分解酵素遺伝子にセルロース分解酵素を菌体外に放出する能力があるかどうかを確かめた。セルロース分解酵素遺伝子を導入した大腸菌ＨＢ１０１株を培養し，菌体内外のセルロース分解酵素の存在場所を調べたが，この遺伝子には今までに知られているような菌体外への放出機構は認められなかった。しかしこの遺伝子は細胞膜構造の一部に変異のある大腸菌ＪＭ１０９株の膜構造に作用して，セルロース分解酵素のみならず大腸菌に存在する菌体内酵素も菌体外に放出させる機能をもつことが明らかとなった。

概要 （英文）

HYDROGEN IS EXPECTED TO BECOME AN IMPORTANT ENERGY SOURCE BECAUSE OF ITS CLEANLINES AND APPLICABILITY FOR FUEL CELLS. RESEARCH HAS BEEN CONDUCTED ON BREEDING ANAEROBIC BACTERIA WHICH HASAN EMPHASIZED ABILITY ON HYDROGEN PRODUCTION FROM CELLULOSE BY USING GENE MANIPULATION METHODS. PREVIOUSLY,WE CLONED TWO CELLULASE GENES FROMCELLULOLYTIC ANAEROBIC BACTERIA CLOSTRIDIUM CELLOBIOPARUM. IN THIS REPORT,WE DESCRIBE THE DISTRIBUTION OF CELLULASE ACTIVITY IN ESCHERICHIA COLI STRAIN HB101 OR JM109 HARBORING THE CELLULASE GENE. E.COLI STRAIN HB101 OR JM109 HARBORING RECOMBINANT PLASMIDS PCMC1 WAS HARBESTED AND THE DESTRIBUTION OF CELLULASE ACTIVITY WAS DETERMINED. E.COLI HB101 CARRYING THE PLASMID PCMC1 (E.COLI HB101(PCMC1)) DID NOT EXCRETE CELLULASE ACTIVITY INTO A PERIPLASMIC FRACTION OR AN EXTRACELLULAR FRACTION. THIS DATA SUGGEST THAT THE PLASMID PCMC1 DO NOT INCLUDE A SIGNAL SEQUENCE FOR EXCRETION. ON THE OTHER HAND E.COLI JM109(PCMC1) EXCRETED MORE THAN HALF OF THE CELLULASE ACTIVITY(57%) INTO A EXTRACELLULAR FRACTION. BETA-LACTAMASE AND MALATE DEHYDROGENASE ACTIVITIES WERE MEASURED AS MARKER ENZYMES IN A PERIPLASM AND CYTOPLASM,RESPECTIVERY. BETA-LACTAMASE AND MDH WERE ALSO FOUND IN THE EXTRACELLULAR FRACTION. IN TIME COURSE EXPERIMENT OF E.COLI JM109(PCMC1),NO LYSIS OF THE CELLS WERE OBSERBED EVEN WHEN THE EXTRACELLULAR CELLULASE ACTIVITIES REACHED MAXIMUM. THIS DATA INDICATED THE RELEASE OF ENZYMES IS NOT CAUSED BY AUTOLYSIS. WHEN E.COLI JM109 CARRYING THE PLASMID WHICH HAD A DELETION AT 0.5KB BGLII-BAMHI FRAGMENT WAS CULTIVATED,ALL OF THREE ENZYME ACTIVITIES (CELLULASE,BETA-LACTAMASE AND MDH) WERE MAINLY DETECTED IN AN INTRACELLULAR FRACTION. THESE DATA SUGGEST THAT THE 0.5KB BGLII-BAMHI FRAGMENT INCLUDES A DNA SEQUENCE WHICH DIRECT TO CHANGE A PERMEABILITY OF THE MEMBRANE OF E.COLI JM109 AND TO EXCRETE BOTH OF PERIPLASMIC AND CYTOPLASMIC ENZYMES INTO A MEDIUM.

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