Characterization of the White-rot Fungus, Phanerochaete carnosa , through Proteomic Methods and Compositional Analysis of Decayed Wood Fibre By Sonam Mahajan A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Department of Chemical Engineering and Applied Chemistry University of Toronto © Copyright by Sonam Mahajan 2011 Characterization of the white-rot fungus, Phanerochaete carnosa , through proteomic methods and compositional analysis of decayed wood fibre Sonam Mahajan Doctorate of Philosophy Department of Chemical Engineering and Applied Chemistry University of Toronto 2011 Abstract Biocatalysts are important tools for harnessing the potential of wood fibres since they can perform specific reactions with low environmental impact. Challenges to bioconversion technologies as applied to wood fibres include low accessibility of plant cell wall polymers and the heterogeneity of plant cell walls, which makes it difficult to predict conversion efficiencies. White-rot fungi are among the most efficient degraders of plant fibre (lignocellulose), capable of degrading cellulose, hemicellulose and lignin. Phanerochaete carnosa is a white-rot fungus that, in contrast to many white-rot fungi that have been studied to date, was isolated almost exclusively from fallen coniferous trees (softwood). While several studies describe the lignocellulolytic activity of the hardwood-degrading, model white-rot fungus Phanerochaete chrysosporium , the lignocellulolytic activity of P. carnosa has not been investigated. ii An underlying hypothesis of this thesis is that P. carnosa encodes enzymes that are particularly well suited for processing softwood fibre, which is an especially recalcitrant feedstock, though a major resource for Canada. Moreover, given the phylogenetic similarity of P. carnosa and P. chrysosporium , it is anticipated that the identification of pertinent enzymes for softwood degradation can be more easily conducted. In particular, this project describes the characterization of P. carnosa in terms of the growth conditions that support lignocellulolytic activity, the effect of enzymes secreted by P. carnosa on the chemistry of softwood feedstocks, and the characterization of the corresponding secretome using proteomic techniques. Through this study, cultivation methods for P. carnosa were established and biochemical assays for protein activity and quantification were developed. Analytical methods, including FTIR and ToF-SIMS were used to characterize wood samples at advancing stages of decay, and revealed preferential degradation of lignin in the early stages of growth on all softwoods analyzed. Finally, an in depth proteomic analysis of the proteins secreted by P. carnosa on spruce and cellulose established that similar sets of enzyme activities are elicited by P. carnosa grown on different lignocellulosic substrates, albeit to different expression levels. iii Acknowledgements I would like to express my deepest gratitude to Dr. Emma Master for accepting me as her PhD student 5 some years ago, in spite of the sufficient lack of background that I brought with me in this demanding field. Over the years, I have acquired technical skills and developed the ability to learn newer information better - it would certainly not have been possible without her kind patience throughout my learning stages. I am very grateful for her constant guidance, intellectually stimulating discussions, understanding of my personal and technical challenges, and encouragement during the trying times. I am also very grateful to Dr. Dragica Jeremic for all her technical assistance, and more importantly, for her friendly guidance throughout the later years of my PhD. It would have been very difficult for me to see the end of my PhD if it were not for her un-ending support. I am thankful to the members of my committee, Dr. Elizabeth Edwards and Dr. Krishna Mahadevan, for the feedback and direction they provided for this project. I am also grateful to Dr. Robyn Goacher for her technical assistance and contribution to the fibre characterization studies, and to Peter Brodersen for his help in the early phases of fibre characterization experiments with ToF-SIMS. I would also like to express my thanks to Dr. Eric Yang from Sunnybrooke for his collaboration through the Proteomic Studies and to Dr. Tony Ung for his assistance in sugar analysis and kind offering of all technical resources whenever required. I will always remember Jacqueline, for our friendly PhD pep-talks and for the technical and moral support in designing the mammoth fibre characterization experiments. Finally, my huge appreciation for all the members of the Master Lab and BioZone for their cooperation, and for accepting me as a relatively inert member of the group, especially during the final stages of my PhD. iv I am very grateful to my parents, Dr. Ravi Mahajan and Dr. Kalpana Mahajan, who inspired me to begin this marathon journey in life. Over the last 5 years, I’ve learnt a significant amount and in spite of the challenges, I would have never taken it up, if it were not for them. My loving thanks to my sister, Samridhi for adding a fresh breathe of non-academic humor to my sometimes humdrum life. I am indebted to my husband, Ateet, for his never-ending patience, gentle encouragement, kind technical assistance and acceptance of all sloppy standards at home. And to all my dearest friends, whose warmth created for me a home away from home, whose company made dull moments bright, and whose smiles, encouraging emails, texts and generous home visits with food, kept me alive through the most trying times…we did it! Finally, my heartfelt gratitude to all my spiritual teachers and mentors, who have carved on my heart determination and faith in the absolute will, the ability to discern the temporary from the eternal, and the desire to serve with perfection… Things that are very difficult to do become easy to execute if one somehow or other simply remembers Lord Caitanya Mahaprabhu. But if one does not remember Him, even easy things become very difficult. To this Lord Caitanya Mahaprabhu I offer my respectful obeisances. Caitanya Caritamrita Adi Lila 14.1 v Table of Contents Table of Contents ......................................................................................................................... vi List of Figures .................................................................................................................................x List of Abbreviations ................................................................................................................... xi Chapter 1 : Overview .....................................................................................................................1 Chapter 2 : Literature Review ......................................................................................................7 2.1 Lignocellulose: A Valuable Resource ...............................................................................7 2.2 Composition and Structure of Plant Cell Walls ..............................................................8 2.3 Bioconversion of Lignocellulose ........................................................................................9 2.4 Lignocellulose-degrading Bacteria ...................................................................................9 2.5 Lignocellulose-degrading Fungi .....................................................................................10 2.5.1 White-Rot Fungi ...................................................................................................11 2.5.2 Brown-Rot Fungi .................................................................................................11 2.6 Lignocellulose Active Enzymes .......................................................................................12 2.6.1 Carbohydrate Active Enzymes ...........................................................................12 2.6.1.1 Cellulases ..............................................................................................................13 2.6.1.2 Hemicellulases ......................................................................................................13 2.6.1.3 Fungal Oxidative Lignin Enzymes .....................................................................14 2.7 Investigative Approaches to Improve Lignocellulose Bioconversion ..........................14 2.7.1 Analytical Characterization of Wood Fibre ......................................................15 2.7.2 Biochemical Characterization of Wood-degrading Fungi ...............................18 Chapter 3 : Effect of Cultivation Conditions on the Expression of Cellulolytic Activity by Phanerochaete species P. chrysosporium and P. carnosa ................................................30 3.1 Abstract .............................................................................................................................30 3.2 Introduction ......................................................................................................................30 vi 3.3 Review of P. chrysosporium Cultivation and Lignocellulolytic Activity .....................32 3.4 Materials and Methods ....................................................................................................41 3.4.1 Microorganism and Materials ............................................................................41 3.4.2 Cultivation Conditions .........................................................................................41