Identification of Cellulase Gene from the Metagenome of Equus burchelli Fecal Samples and Functional Characterization of a Novel Bifunctional Cellulolytic Enzyme

摘要

The metagenomic approach has been used successfully to isolate novel biocatalyst 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 recombinant protein showed an activity of 5.23 U with specific activity of 6.8 U mg−1 protein with the substrate avicel, while when CMC was used, an activity of 3.0 U with a specific activity of 4.2 U mg−1 protein was achieved. Its optimum pH was determined to be 7.0 and optimum temperature of 35°C.