Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases.

ABSTRACT

A metagenomic cosmid library was prepared in Escherichia coli from DNA extracted from the contents of rabbit cecum and screened for cellulase activities. Eleven independent clones expressing cellulase activities (four endo-beta-1,4-glucanases and seven beta-glucosidases) were isolated. Subcloning and sequencing analysis of these clones identified 11 cellulase genes; the encoded products of which shared less than 50% identities and 70% similarities to cellulases in the databases. All four endo-beta-1,4-glucanases and all seven beta-glucosidases, respectively, belonged to glycosyl hydrolase family 5 (GHF 5) and family 3 (GHF 3) and formed two separate branches in the phylogenetic tree. Ten of the 11 cloned cellulases exhibited highest activities at pH 5.5 approximately 7.0 and 40 approximately 55 degrees C, a condition similar to that in the rabbit cecum. All the four endo-beta-1,4-glucanases could hydrolyze a wide range of beta-1,4-, beta-1,4/beta-1,3- or beta-1,3/beta-1,6-linked polysaccharides. One endo-beta-1, 4-glucanase gene, umcel5G, was overexpressed in E. coli, and the purified recombinant enzyme was characterized in detail. The enzymes cloned in this work represented at least some of the cellulases operating efficiently in the rabbit cecum. This work provides the first snapshot on the cellulases produced by bacteria in rabbit cecum.