|Willis, Jonathan -|
|Oppert, Cris -|
|Klingeman, William -|
|Jurat-Fuentes, Juan -|
Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2010
Publication Date: January 14, 2011
Repository URL: http://hdl.handle.net/10113/48903
Citation: Willis, J.D., Oppert, B.S., Oppert, C., Klingeman, W.E., Jurat-Fuentes, J.L. 2011. Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae). Journal of Insect Physiology. 57(2):300-306. Interpretive Summary: Cellulases are enzymes that can break down plant material. Although cellulases in insects were thought to occur through microbial symbionts, we now know that insects also can make their own cellulase enzymes. In studying the genome sequences of the red flour beetle, we found a gene that encoded a putative cellulase. We used molecular procedures to clone the gene and express the protein. We find that the enzyme has characteristics of a particular group of enzymes called endoglucanases and has highest activity at extremely high pH. Since processing plant materials at high pH may be more efficient, this beetle enzyme may have applications biofuels refineries.
Technical Abstract: The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-ß-1,4-glucanase we named TcEG1 (T. castaneum EndoGlucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1,356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a mutation (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5 kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting and cellulose activity gels. The expressed protein was used to characterize TcEG1 enzymatic activity against diverse cellulose substrates to determine its specificity and stability. Our data support TcEG1 as a novel endo-ß-1,4-glucanase displaying enzymatic activity at alkaline pH, the first characterization of a cellulase enzyme from an insect genome.