Metagenomics of un-culturable bacteria in cow rumen: Construction of cel9E–xyn10A fusion gene by site-directed mutagenesis

Abstract

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. Genetic engineering facilitates the artificial fusion of genes that encode functional proteins into a single open reading frame. The cloning of the cellulase and xylanase genes from the cow rumen metagenome resulted in the construction of a bifunctional fusion gene via site-directed mutagenesis for further specific industrial processes. The metagenome of cow rumen bacteria was the source of a gene that encodes an extracellular β-glucanase for cloning and expression in Escherichia coli DH5α. The cellulase (cel9E) gene of un-culturable rumen bacteria existed in tandem with the xylanase (xyn10A) gene. The genes were 2268bp and 1.578bp and encoded 756- and 526-aa proteins, respectively. BLAST analyses and domain predictions assigned Cel9E and Xyn10A to glycosyl hydrolase families 9 and 10. The molecular weight of the individual proteins Cel9E and Xyn10A were estimated to be approximately 76.0kDa and 56.0kDa by CMC–SDS-PAGE and OSX–SDS-PAGE, respectively. The 3909bp cel9E–xyn10A fusion gene encoded a 1303-amino acid residue protein with a molecular weight of approximately 137.0kDa according to CMC/OSX–SDS-PAGE. The maximum cellulase and xylanase activities from the fusion protein Cel9E–Xyn10A were observed at pH 6.0 and pH 8.0, respectively. The optimal temperature for the bifunctional enzyme was found to be 50°C. The improved catalytic efficiency of the Cel9E–Xyn10A for the cellulase and xylanase activity was equivalent to 1.47- and 2.21-fold of the parental efficiency. We report the presence of the cel9E gene in tandem with the xyn10A gene in the metagenome of un-culturable cow rumen bacteria. The construction, expression and characterization of the cel9E–xyn10A bifunctional gene fusion obtained by site-directed mutagenesis are also reported.