Isolation and characterization of a non-specific endoglucanase from a metagenomic library of goat rumen.

Abstract

A cellulase gene (cel28a) was isolated from a rumen microbial metagenome library of goat rumen microorganisms, cloned into E. coli, and expressed in active form. The gene has a length of 1596 bp obtained using a genome walking Kit and encodes a protein of 509 amino acids with a calculated MW of 55 kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolase family 5 (GH5). The expressed protein showed activity toward carboxymethylcellulose (CMC) and xylan, suggesting non-specific endoglucanase activity. The optimal conditions for endoglucanase and xylanase activities were 50 °C and pH 5.0. The metal ions (Ca(2+), Fe(2+), Mn(2+) and Co(2+)) stimulated the cellulase activity of cel28a, while the other metal ions and chemicals (Ni(2+), Mg(2+), Zn(2+), Cu(2+), SDS and EDTA) inhibited the cellulase activity. Further examination of substrate preference showed a higher activity with CMC, oat spelt xylan and birchwood xylan than with filter paper and microcrystalline cellulose, again suggesting that the protein was an endoglucanase with xylanase activity.