Cloning of the Ruminococcus albus cel5D and cel9A Genes Encoding Dockerin Module-containing Endoglucanases and Expression of cel5D in Escherichia coli
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概要
- 論文の詳細を見る
An EcoRI chromosomal DNA fragment of Ruminococcus albus F-40 that conferred endoglucanase activity on Escherichia coli was cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5D and cel9A respectively. The cel5D gene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The cel9A gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E. coli. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 μmol/min/mg for carboxymethycellulose.
- 社団法人 日本農芸化学会の論文
- 2004-07-23
著者
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Ohmiya K
Graduate School Of Bioresources Mie Univ.
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Ohmiya Kunio
Faculty Of Bioresources Mie University
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KIMURA Tetsuya
Faculty of Bioresources, Mie University
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Kajino T
Special Research Laboratory Ii Toyota Central R&d Laboratories Inc.
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Kimura Tetsuya
National Center For Seeds And Seedlings
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Karita Shuichi
Faculty of Bioresources, Mie University
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SAKKA Kazuo
Faculty of Bioresources, Mie University
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TAGUCHI Hidenori
Faculty of Bioresources, Mie University
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HAGIWARA Daisuke
Faculty of Bioresources, Mie University
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GENMA Tsutomu
Faculty of Bioresources, Mie University
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Sakka Kazuo
Faculty Of Bioresources Mie University
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Genma Tsutomu
Faculty Of Bioresources Mie University
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Karita Shuichi
Faculty Of Bioresources Mie University
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Kimura Tetsuya
Graduate School Of Bioresources Mie University
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HAGIWARA Daisuke
New Industry Creation Hatchery Center, Tohoku University
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Hagiwara Daisuke
New Industry Creation Hatchery Center Tohoku University
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Taguchi Hidenori
Faculty Of Bioresources Mie University
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Kimura Tetsuya
Faculty Of Bioresources Mie University
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