| Title |
Screening and Characterization of a Cellulase Gene from the Gut Microflora of Abalone Using Metagenomic Library |
| Author |
Duwoon Kim1, Se-Na Kim2,3, Keun Sik Baik2, Seong Chan Park2, Chae Hong Lim2, Jong-Oh Kim3, Tai-Sun Shin3, Myung-Joo Oh3, and Chi Nam Seong2* |
| Address |
1Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea, 2Department of Biology, Sunchon National University, Suncheon 540-742, Republic of Korea, 3Korea Basic Science Institute, Suncheon Center, Suncheon 540-742, Republic of Korea |
| Bibliography |
Journal of Microbiology, 49(1),141-145, 2011,
|
| DOI |
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| Key Words |
abalone, cellulase, metagenomic library, gut microflora |
| Abstract |
A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAM11E10). A shotgun clone library was constructed using the positive clone (pAM11E10) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAM11L9). The pAM11L9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAM11) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus
12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAM11) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAM11 were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose. |
