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University of Florida Thesis Or Dissertation Formatting EXPLORATION AND OPTIMIZATION OF THREE ENZYMATIC ROUTES TO CONVERT LINOLEIC ACID INTO N-HEXANAL By ERICA AMATO SIMMONS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Erica Simmons To my loving parents, Pasquale and Denise Amato and husband, Aaron Simmons ACKNOWLEDGMENTS I would first like to thank Dr. Jon Stewart for his continuing support and encouragement throughout my Ph.D. work. His guidance has been essential, especially at times when I just wanted to quit the project due to constant failures. He motivated me to keep going and to see the project through to completion, without which I would have never been able to ultimately find a method to convert linoleic acid into n-hexanal. I also need to mention the contributions from other faculty members such as Dr. Horenstein, Dr. Butcher and Dr. Bruner for always being available for advice regarding my project and for allowing me to use lab equipment when it was broken in my laboratory. Much of the reaction progress mentioned throughout this work would not have been possible without the help of their labs. I need to specially thank my undergraduate mentee, Olivia Toudjarov, who despite her hectic schedule would come to the laboratory to do research. I would also like to thank the various members of my research group for always being willing to help when asked. Next I would like to thank the people who got me through this graduate school program including Mayra Rostagno, Jessica Cash, Sandy Guntaka, and Kim Stewart. They were always willing to give me suggestions when I didn’t know where to go next in my research. In addition, they were crucial in making sure I didn’t quit my project when it seemed impossible to finish. They encouraged me to try other directions and to take breaks to clear my mind before returning to the project. It is always encouraging to know that various disciplines can come together to approach a project from a new perspective and think of new strategies despite the fact that it is not their strength. Finally, I would like to thank my family for constant encouragement and support when hard times came up. Being over a thousand miles away from them was not easy throughout this Ph.D. program, but we always found ways of seeing each other and were in constant 4 communication. At times, they were the motivation I needed to go into lab the next day and push through the constant failures that accumulated. I need to give a special thanks to my husband, Aaron Simmons, for putting up with me when my research stressed me out and became frustrating. He managed to always find ways of making me forget about work and to relax. His love and support always push me to be a better person and ultimately to complete this Ph.D. program. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................11 ABSTRACT ...................................................................................................................................15 CHAPTER 1 INTRODUCTION ..................................................................................................................16 Short Chain Aldehydes and Ketones ......................................................................................16 European Union Regulations on Natural Flavors ...................................................................18 Current n-Hexanal Synthetic Routes ......................................................................................18 Chemical Synthesis Approaches .....................................................................................18 Natural Sources ...............................................................................................................20 Bioprocesses ....................................................................................................................22 New Strategies for Producing n-Hexanal from Linoleic Acid ...............................................24 Castor Oil Pyrolysis ................................................................................................................26 Carotenoid Cleavage Dioxygenases .......................................................................................27 Identification and Characterization .................................................................................27 Carotenoid cleavage dioxygenase 1 (EC: 1.14.99.n4) .............................................29 Carotenoid cleavage dioxygenases 7 (EC:1.13.11.68) and 8 (EC:1.13.11.691) ......31 CCD Enzyme Structure ...................................................................................................33 Mechanism ......................................................................................................................36 Mutagenesis Studies ........................................................................................................39 Applications of CCD1 .....................................................................................................39 Lipoxygenases ........................................................................................................................41 Identification and Characterization .................................................................................41 Enzyme Structure ............................................................................................................43 Mechanism ......................................................................................................................46 Lineolate 13-Hydratase ...........................................................................................................48 Identification and Characterization .................................................................................48 Enzyme Structure ............................................................................................................51 Mechanism ......................................................................................................................54 2 USING CAROTENOID CLEAVAGE DIOXYGENASES TO CLEAVE LINOLEIC ACID INTO N-HEXANAL ....................................................................................................56 Background .............................................................................................................................56 Experimental Strategy ............................................................................................................56 n-Hexanal Quantitation ...................................................................................................56 CCD Production and Isolation .........................................................................................57 6 Cleaving Linoleic Acid to n-Hexanal Using CCD1 Enzymes ........................................58 Experimental Procedures ........................................................................................................58 Light Scattering Experiments ..........................................................................................58 n-Hexanal Quantitation ...................................................................................................59 1. Solid phase micro extraction (SPME) ..................................................................59 GC/MS analysis using EDA2_MTH ........................................................................59 2. 2,4-Dinitrophenylhydrazine (DNPH) derivitization .............................................60 3. Solvent extraction using d12-n-hexanal .................................................................61 Resin recoveries .......................................................................................................61 Linoleic Acid Quantitation ..............................................................................................61 FAME derivitization ................................................................................................61 MSTFA derivitization ..............................................................................................62 GC/MS analysis: EA1_METH .................................................................................62 ZmCCD1 Expression .......................................................................................................63 Construction of pEA1 ......................................................................................................63 Construction of pEA2 ......................................................................................................64 Construction of pEA3 ......................................................................................................64 AtCCD1 Expression .........................................................................................................64 Acetylation of AtCCD1 ...................................................................................................65 CLEA Preparation ...........................................................................................................65
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