Identification and Characterization of Novel Cellulases from <em>Dissosteira carolina</em> (Orthoptera: Acrididae) and Molecular Cloning and Expression of an Endo-beta-1,4-Glucanase from <em>Tribolium castaneum</em> (Coleoptera: Tenebrionidae)

• Main Author: Willis, Jonathan Duran
• Format: Others
• Published: Trace: Tennessee Research and Creative Exchange 2009
• Subjects: Entomology
• Online Access: http://trace.tennessee.edu/utk_gradthes/573

Cellulosic ethanol holds great potential as biofuel due to its sustainability and renewability, yet recalcitrance of cellulosic feedstocks prevents cost-efficient ethanol production. Enzymatic catalysis of lignocellulosic biomass has the greatest biotechnological potential for cost reductions to the production process. Even though numerous cellulolytic enzymes have been identified in bacteria, plant, and fungi, insects remain as a fairly unexplored prospecting resource. Many insects, either via endogenously or symbiotically derived enzymes, use cellulose as substrate for their energetic needs. Novel cellulases from insects may have the potential to be more efficient than alternative enzymes in the conversion of cellulose to fermentable sugars due to their optimized activity in the highly reducing and extreme pH conditions found in some insect digestive systems. In this work we present data characterizing cellulolytic activity in the grasshopper Dissosteira carolina L. (Orthoptera: Acrididae) and the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae). After a screening for cellulolytic activity in insect populations from the East Tennessee region, D. carolina was selected due to relatively high cellulolytic activity compared to documented effective insect cellulolytic species. Cellulolytic activity in digestive fluids from gut and head from juvenile and adult stages of D. carolina was measured and an active cellulolytic protein profile demonstrated comparable activities amongst life stages. Partial protein sequences that match those identified from insect and microbial cellulases were obtained from purified 43-kDa and 45-kDa cellulases from D. carolina head digestive fluids. Although unsuccessful, attempts were made to purify and clone these enzymes for recombinant expression. Our research on D. carolina is the first report on the purification of endoglucanase activity in a grasshopper species. Availability of the T. castaneum genome allowed for homology searches using reported insect cellulases to identify a predicted cellulase. We cloned the full-length cDNA for this enzyme and named it TcEG1 (for T. castaneum endo-glucanase-1). TcEG1 was heterologously expressed in bacterial and insect cell culture systems and its activity against cellulose substrates and thermostability measured. Cloning of a cellulase gene from T. castaneum adds to the collection of reported insect cellulases and demonstrates the advantage of using genomic resources for protein discovery.