Identification of Cellulase Gene from the Metagenome of Equus burchelli Fecal Samples and Functional Characterization of a Novel Bifunctional Cellulolytic Enzyme Matam Chandrasekharaiah & Appoothy Thulasi & Madiajagan Bagath & Duvvuri Prasanna Kumar & Sunil Singh Santosh & Chenniappan Palanivel & Vazhakkala Lyju Jose & Koratokare Thirumalachar Sampath Received: 13 June 2011 / Accepted: 28 March 2012 / Published online: 20 April 2012 # Springer Science+Business Media, LLC 2012 Abstract The metagenomic approach has been used successfully to isolate novel bio- catalyst gene from uncultured microorganisms. The gene encoding exo-1,4-β-glucanase avicelase was amplified from the metagenome of the Equus burchelli fecal sample and cloned. The gene was found to be of 1,007 bp of nucleotide which encodes a protein of 318 amino acids with a calculated MW of 36 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolases 6 superfamily. The expressed protein was active towards the substrates avicel and carboxymethyl cellulose, indicating that it has bifunctional cellulolytic enzyme activity. The recombi- nant protein showed an activity of 5.23 U with specific activity of 6.8 Umg -1 protein with the substrate avicel, while when CMC was used, an activity of 3.0 U with a specific activity of 4.2 Umg -1 protein was achieved. Its optimum pH was determined to be 7.0 and optimum temperature of 35°C. Keywords Exo-1,4-β-glucanase . Avicelase . Metagenome . Enzyme activity . Cloning Introduction Cellulose is the most abundant carbohydrate polymer in the biosphere. It is a recalcitrant polymer that exists mainly in plant and humic matter with other polymers such as hemicel- lulose and lignin. Cellulase is responsible for the hydrolytic cleavage of β-glycosidic bonds in cellulose. They can be broadly classified in three classes based on their mode of action, Appl Biochem Biotechnol (2012) 167:132–141 DOI 10.1007/s12010-012-9660-5 M. Chandrasekharaiah (*) : A. Thulasi : M. Bagath : D. P. Kumar : S. S. Santosh : C. Palanivel : V. L. Jose : K. T. Sampath Rumen Microbiology Laboratory, Animal Nutrition Division, National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, Karnataka 560030, India e-mail: chandrasekhariah.naip@gmail.com