A comparative study of the structural and physicochemical properties of the major proteins from Camelina sativa (L.) Crantz and Brassica napus L. A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon By Suneru P. P. Ussetti Mohottalalage 2016 © Copyright Suneru Ussetti Mohottalalage, September 2016. All rights reserved i PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying, publication, or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon, Saskatchewan, S7N 5A8 Canada i ABSTRACT Camelina sativa (L.) Crantz is a new industrial oilseed crop suitable for the Canadian prairies and which shows potential benefits for the Canadian bio-economy. This study was carried out to identify the major proteins of the oil-free residue (meal) of C. sativa (camelina) while investigating their structural and physicochemical properties. Canola (Brassica napus L.) was used as the control in the study. Camelina seeds were treated with Viscozyme® (0.1 mL/g) to remove mucilage. The mucilage free meal contained 51.3% protein (dwb, %N×6.25) which was greater than in canola. Both camelina and canola meals shared similar profiles for polypeptides and amino acids. At acidic pHs, canola meal had higher soluble protein content than did camelina meal, but the opposite was observed when the pH moved toward alkaline. A pH of 4.5 identified as the apparent isoelectric point (pI) of the protein from these two meals, which is presumably a cruciferin-napin complex. The 11S and 2S proteins of both seed types were isolated and purified using liquid chromatography. The purified 11S protein from camelina and canola contained predominantly cruciferin with minor contamination with non-targeted storage proteins. Of the non-cruciferin contamination of camelina, vicilin (7S) found to be abundant. The purified 2S protein from camelina contained napin and a noticeable amount of late embryogenesis abundant (LEA) protein, whereas non-napin contamination was minor in canola. In camelina, cruciferin, napin and vicilin expressed from eleven, four and six genes, respectively, were identified. The oil body proteins were also isolated and several isoforms of oleosin were found in camelina and canola, whereas putative isoforms of caleosin and steroleosin were found only in camelina. The structural and physicochemical properties of cruciferin and napin were studied in response to changing pH and temperature. The predominance of -structure and α-helix content in the 2˚ structure of cruciferin and napin, respectively, was confirmed for both camelina and canola. Cruciferin from camelina and canola exhibited acid-induced structural unfolding at the 3˚ structure level. Cruciferin was not completely unfolded and assumed an intermediate state, plausibly a molten globule. Napin structure was not as sensitive as cruciferin to changing medium pH or an increase in temperature. Cruciferin exhibited high thermal stability (>80˚C) at neutral and alkaline pH, whereas the opposite was observed at acidic pH. Results showed that the cruciferin and napin responded differently to changing pH and temperature. Therefore, conditions of oil extraction and protein recovery from meal may affect these two storage proteins differently. ii ACKNOWLEDGEMENTS I would like to express my humble gratitude to my supervisors Drs. Janitha Wanasundara and Robert Tyler for their continued guidance and encouragement, constructive criticisms, and valuable advices provided throughout the M.Sc. Program. I would like to thank my advisory committee members, Drs. Dwayne Hegedus and Phyllis Shand for the continuous support and expertise provided to progress throughout the project and complete the thesis. Also I sincerely thank Dr. Xiao Qiu, and Dr. Darren Korber who are the current and former chairs, respectively, for my graduate advisory committee for their suggestions and advices provided in many aspects throughout the programme to make this research a success. I would like to extend my thanks to Dr. Rex Newkirk for serving as the external examiner. I am grateful to the research and administration staff of the Agriculture and Agri-Food Canada (AAFC), Saskatoon Research and Development Center for the opportunity given to work under the Research Affiliate Program (RAP), and providing facilities to conduct this research project. I would like to especially thank Tara McIntosh for the immense support and expertise provided to conduct the experiments. I take this opportunity to thank Cathy Coutu, Myrtle Harrington, Dan Southerland, Dan Hupka, Debbie Anderson, Doug Baldwin and Simarpreet Singh for supporting me in numerous ways to succeed the goals of my thesis research. Also, I am grateful to Dr. Kevin Falk for providing field grown camelina seeds required for this study. I am thankful to Agriculture and Agri-Food Canada research project “Camelina-an integrated Industrial Oilseed Crop Platform for Canada (RBPI 2535) and University of Saskatchewan, Global Institute of Food Security (GIFS) project “Developing Camelina as a Modern Crop Platform” for the funding support for my research and graduate program. I would like to thank my wife Shwetha Jayani Liyange, my parents Mr. Clinton Susantha Perera and Mrs. Vineetha Vithanage, and my brother Vidyu Suyama Perera for being in my side for the every step of this journey. I especially thank Dr. Manjula Bandara and Mrs. Chandara Bandara from Saskatoon for their continuous encouragement and support. Finally, I thank my fellow graduate students in the department of Food and Bioproduct Sciences and all my friends who were there to support me whenever I was in need. iii TABLE OF CONTENTS PERMISSION TO USE ....................................................................................................... i ABSTRACT ........................................................................................................................ ii ACKNOLEDGEMENTS ................................................................................................... iii TABLE OF CONTENT ..................................................................................................... iv LIST OF FIGURES ......................................................................................................... viii LIST OF TABLES .............................................................................................................. x LIST OF ABBRIVIATIONS ............................................................................................. xi 1. INTRODUCTION .......................................................................................................... 1 1.1 Hypotheses ................................................................................................................ 3 1.2 Objectives .................................................................................................................. 3 2. LITERATURE REVIEW ............................................................................................... 4 2.1 Biodiesel and oilseeds ............................................................................................... 4 2.2 Brassicaceae species as industrial oilseed crops for Canada .................................... 6 2.3 Camelina as an industrial oilseed crop for Canada ................................................... 9 2.3.1 Agronomic characteristics .................................................................................. 9 2.3.2 Genetics and breeding ........................................................................................ 9 2.3.3 Chemical characteristics of the seed and meal…………………………………13 2.4 Camelina meal and the current situation ................................................................. 16 2.5 Overview of seed protein ........................................................................................ 18 2.5.1 Types of seed storage proteins.......................................................................... 18 2.5.2 Oil body proteins .............................................................................................. 19 iv 2.6 Storage proteins of Brassicaceae oilseeds .............................................................. 20 2.6.1 Cruciferin……………………………………………………………………...20 2.6.2 Napin………………………………………………………………………….23 2.7 Summary ................................................................................................................. 25 3. MATERIALS