Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

• Main Authors: Gong Xia, Gruninger Robert J, Qi Meng, Paterson Lyn, Forster Robert J, Teather Ron M, McAllister Tim A
• Format: Article
• Language: English
• Published: BMC 2012-10-01
• Series: BMC Research Notes
• Subjects: Endoglucanase; Ruminal microorganisms; BAC library; Dairy cow
• Online Access: http://www.biomedcentral.com/1756-0500/5/566

<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 Mn²⁺ and dramatically reduced by Fe³⁺ or Cu²⁺. 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>