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Open Access Highly Accessed Research article

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

Xia Gong12, Robert J Gruninger1, Meng Qi1, Lyn Paterson1, Robert J Forster1, Ron M Teather1 and Tim A McAllister1*

Author Affiliations

1 Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, T1J 4B1, Canada

2 Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China

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BMC Research Notes 2012, 5:566  doi:10.1186/1756-0500-5-566

Published: 13 October 2012

Abstract

Background

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.

Results

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 Cel14b22, was expressed at a high level in Escherichia coli 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.

Conclusion

The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

Keywords:
Endoglucanase; Ruminal microorganisms; BAC library; Dairy cow