Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae)

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-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (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. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.

Figure optionsDownload as PowerPoint slideResearch highlights▶ The Tribolium castaneum genome glean 15370 shows homology to endoglucanases. ▶ This gene encodes a 50-kDa protein, named TcEG1 (T. castaneum endoglucanase 1). ▶ TcEG1 is the first glycosyl hydrolase family 9 cellulase reported for Coleoptera. ▶ TcEG1 displays endoglucanase activity thermostable up to 60 °C. ▶ TcEG1 cellulase activity is highest at alkaline pH.