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Functional Characterization of an Endoglucanase from <I>Tribolium

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Functional Characterization of an Endoglucanase from <I>Tribolium University of Tennessee, Knoxville Trace: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2011 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) Derek Mychel Shirley [email protected] Recommended Citation 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. https://trace.tennessee.edu/utk_gradthes/1097 This Thesis is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Derek Mychel Shirley entitled "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)." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Entomology and Plant Pathology. Juan Luis Jurat-Fuentes, Major Professor We have read this thesis and recommend its acceptance: William E. Klingeman, Todd B. Reynolds Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) 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) A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Derek Mychel Shirley December 2011 ii Copyright © 2011 by Derek Mychel Shirley All rights reserved. iii Dedication This work is dedicated to my parents, Pam and Terry, and my brother Drew who supported me, and stood by me. Without you none of this would have ever been possible. iv Acknowledgements I gratefully acknowledge Dr. Juan-Luis Jurat-Fuentes for his financial support and leadership. Most importantly I would like to thank him for the immense amount of patience needed to complete my studies. I would also like to thank the members of my committee Dr. William Klingeman, Dr. Todd Reynolds for their invaluable advice and input throughout this entire process. Additionally I would like to thank all of the members of the Jurat-Fuentes lab: Dr. Cris Oppert, Jerreme Jackson, Anais Castagnola, Siva Jakka, and Victoria Knight; for creating an amazing environment for both learning and expanding on ideas. You all made it a joy each and every day. Finally I would like to acknowledge Patrick Stanley and Eric Snell (Snell Scientific, LLC) and George Keeney (Ohio State University) for supplying the colonies of T. domestica as well as the guidance and insight into rearing the insects, and the Southeastern Sun Grant Center for the financial support to pursue the further investigation of novel insect catalysts in the use of biofuel production. v Abstract 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. vi 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. vii Table of Contents Chapter 1 The evolution of endogenous cellulases in insects............................................ 1 Introduction ..................................................................................................................... 2 Cellulose and cellulases .................................................................................................. 4 Insect cellulases .............................................................................................................. 6 The evolution of insect cellulases ................................................................................... 8 Phylogenetic analyses of GHF9 insect cellulases ......................................................... 10 Conclusion .................................................................................................................... 18 Chapter 2 Functional characterization of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae ............................................................................. 20 Introduction ................................................................................................................... 22 Materials and Methods .................................................................................................. 25 Cloning of insect cellulase into expression plasmid p426-GPD ............................... 25 Transformation of yeast with expression plasmid p426-GPD-TcEG1 ..................... 26 Expression and partial purification of TcEG1 from yeast cultures ........................... 28 Protein electrophoresis and zymography .................................................................. 28 In-solution cellulolytic activity assay ....................................................................... 30 pH and thermal stability ............................................................................................ 32 Statistics .................................................................................................................... 33 Results ........................................................................................................................... 33 Expression of TcEG1 in yeast ................................................................................... 33 Quantitative characterization of TcEG1 activity ...................................................... 36 Stability of TcEG1 cellulolytic activity with varying temperature and pH .............. 38 Discussion ..................................................................................................................... 41 Chapter 3 Characterization of cellulolytic activity in the digestive fluids of Thermobia domestica (Zygentoma: Lepismatidae) ............................................................................. 45 Abstract ......................................................................................................................... 46 Introduction ................................................................................................................... 47 Materials and Methods .................................................................................................. 50 Firebrat rearing and dissection .................................................................................. 50 In-Solution cellulolytic activity assays ..................................................................... 51 Enzymatic activity with varying pH ........................................................................
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