Functional characterization of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae and characterization of cellulolytic activity in the digestive fluids of Thermobia domestica (Zygentoma: Lepismatidae)
Date of Award
Master of Science
Entomology and Plant Pathology
Juan Luis Jurat-Fuentes
William E. Klingeman, Todd B. Reynolds
Cellulosic ethanol has the potential to be a significant biofuel due to its sustainability and high energy yield. However it is the recalcitrance of cellulose that presents a costly road block in the economic feasibility of cellulosic ethanol production. The enzymatic degradation of lignocellulosic biomass has the greatest potential for reducing the production costs of this process. While many commercially viable cellulases have been identified in bacteria and fungi, insects remain a relatively untapped source of these enzymes. In fact recent studies have shown that insects produce highly active endogenous cellulases capable of completely hydrolyzing cellulose to glucose, without the presence of multiple enzymatic subunits. Discovering these novel cellulases from insects may help to develop an extremely efficient method of converting cellulose to fermentable sugars, while drastically reducing the energetic input needed with the current batch of commercially available enzymes.
In this work, we present data on the first report of expression of an insect-derived cellulase in a yeast heterologous system. Our goal was to adapt Saccharomyces cerevisiae yeast within a novel functional assay to advance high throughput identification of functional cellulases from genomic insect resources. As a model cellulase, we expressed the full-length cDNA encoding the TcEG1 endoglucanase from the red flour beetle (Tribolium castaneum) under the control of the glyceraldehyde-3 phosphate dehydrogenase (GPD) promoter. TcEG1 was heterologously expressed in this yeast system and its activity against cellulose substrates as well as its pH and thermal stability measured.
Additionally, we examined and characterized the cellulolytic activity in the household paper pest Thermobia domestica (Zygentoma: Lepismatidae). Initial screening of T. domestica showed relatively high cellulolytic activity compared to other documented insect species. Cellulolytic activity from gut and head-derived digestive fluids was measured, and an active cellulolytic protein profile was established. Partial protein sequences that matched those identified from insect, microbial, and other invertebrate cellulases were obtained from partially purified cellulase samples following protein separation. Our research on T. domestica represents the first report on the purification and characterization of endoglucanase activity in a species of Lepismatidae.
Shirley, Derek Mychel, "Functional characterization of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae and characterization of cellulolytic activity in the digestive fluids of Thermobia domestica (Zygentoma: Lepismatidae). " Master's Thesis, University of Tennessee, 2011.