Screening and characterization of a novel ruminal cellulase gene (Umcel-1) from a metagenomic library of gayal (Bos frontalis)

Abstract

Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38 400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase (GenBank accession no. YP_004310852.1) from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coli BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.

Keywords

• gayal
• ruminal microorganism
• fosmid library
• Umcel-1 gene
• gene expression
• cellulase activity