Characterisation of Two Bifunctional Cellulase-Xylanase Enzymes Isolated from a Bovine Rumen Metagenome Library

Ruminant digestive tract microbes hydrolyse plant biomass, and the application of metagenomic techniques can provide good coverage of their glycosyl hydrolase enzymes. A metagenomic library of circa 70,000 fosmids was constructed from bacterial DNA isolated from bovine rumen and subsequently screened for cellulose hydrolysing activities on a CMC agar medium. Two clones were selected based on large clearance zones on the CMC agar plates. Following nucleotide sequencing, translational analysis and homology searches, two cellulase encoding genes (cel5A and cel5B) belonging to the glycosyl hydrolyse family 5 were identified. Both genes encoded pre-proteins of about 62 kDa, containing signal leader peptides which could be cleaved to form mature proteins of about 60 kDa. Biochemical characterisation revealed that both enzymes showed alkaline pH optima of 9.0 and the temperature optima of 65 A degrees C. Substrate specificity profiling of the two enzymes using 1,4-beta-d-cello- and xylo-oligosaccharides revealed preference for longer oligosaccharides (n a parts per thousand yen 3) for both enzymes, suggesting that they are endo-cellulases/xylanases. The bifunctional properties of the two identified enzymes render them potentially useful in degrading the beta-1,4 bonds of both the cellulose and hemicellulose polymers.