Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene
<p>Abstract</p> <p>Background</p> <p>Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzym...
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BMC
2012-10-01
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| Series: | BMC Research Notes |
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| Online Access: | http://www.biomedcentral.com/1756-0500/5/566 |
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| author | Gong Xia Gruninger Robert J Qi Meng Paterson Lyn Forster Robert J Teather Ron M McAllister Tim A |
| author_facet | Gong Xia Gruninger Robert J Qi Meng Paterson Lyn Forster Robert J Teather Ron M McAllister Tim A |
| author_sort | Gong Xia |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow.</p> <p>Results</p> <p>Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated <it>Cel14b22</it>, was expressed at a high level in <it>Escherichia coli</it> and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn<sup>2+</sup> and dramatically reduced by Fe<sup>3+</sup> or Cu<sup>2+</sup>. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum.</p> <p>Conclusion</p> <p>The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.</p> |
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| spelling | doaj.art-6da2511bbc894b8cb7e1127ce3da8d802022-12-22T01:45:07ZengBMCBMC Research Notes1756-05002012-10-015156610.1186/1756-0500-5-566Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase geneGong XiaGruninger Robert JQi MengPaterson LynForster Robert JTeather Ron MMcAllister Tim A<p>Abstract</p> <p>Background</p> <p>Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow.</p> <p>Results</p> <p>Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated <it>Cel14b22</it>, was expressed at a high level in <it>Escherichia coli</it> and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn<sup>2+</sup> and dramatically reduced by Fe<sup>3+</sup> or Cu<sup>2+</sup>. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum.</p> <p>Conclusion</p> <p>The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.</p>http://www.biomedcentral.com/1756-0500/5/566EndoglucanaseRuminal microorganismsBAC libraryDairy cow |
| spellingShingle | Gong Xia Gruninger Robert J Qi Meng Paterson Lyn Forster Robert J Teather Ron M McAllister Tim A Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene BMC Research Notes Endoglucanase Ruminal microorganisms BAC library Dairy cow |
| title | Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene |
| title_full | Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene |
| title_fullStr | Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene |
| title_full_unstemmed | Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene |
| title_short | Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene |
| title_sort | cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene |
| topic | Endoglucanase Ruminal microorganisms BAC library Dairy cow |
| url | http://www.biomedcentral.com/1756-0500/5/566 |
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