Biocatalysis of Soybean 15-Lipoxygenase for the Production of Lipid Mediators of Inflammation by Eleanor Polly Dobson B. Forensic Sc. (Hons) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Deakin University April, 2016 “Arriving at one goal is the starting point to another.” −John Dewey CONTENTS Abstract ..............................................................................................................................vi Acknowledgements .............................................................................................................. x List of Publications .............................................................................................................xi CHAPTER I......................................................................................................................... 1 1. General Introduction .................................................................................................... 2 1.1 Specialised pro-resolving lipid mediators .................................................................... 2 1.2 Enzyme biocatalysts: applications and potential .......................................................... 4 1.3 Research objectives .................................................................................................... 6 CHAPTER II ....................................................................................................................... 9 2. Literature Review ...................................................................................................... 10 2.1 Omega-3 and omega-6 fatty acids ............................................................................. 10 2.2 Specialised Pro-resolving Mediators (SPM) .............................................................. 12 2.3 Pharmaceutical/nutraceutical potential of SPMs ........................................................ 18 2.4 Classification of lipoxygenase .................................................................................. 19 2.5 Soybean 15-lipoxygenase: Catalysis, substrates and specificity ................................. 22 2.6 Production of hydro(pero)xylated compounds from LC-PUFA.................................. 36 2.7 Dependence of regiospecificity and stereospecificity on experimental conditions ...... 51 2.8 Anaerobic reaction of 15-sLOX and production of carbonyl compounds ................... 53 2.9 Conclusion ............................................................................................................... 57 CHAPTER III .................................................................................................................... 58 3. Solvent-Induced 7R-Dioxygenase Activity of Soybean 15-Lipoxygenase-1 in the Formation of Omega-3 DPA-Derived Resolvin Analogs..................................................... 59 3.1 Introduction .............................................................................................................. 59 3.2 Materials and Methods ............................................................................................. 63 3.2.1 Materials ........................................................................................................... 63 3.2.2 Lipoxygenase-catalysed reaction – general protocol .......................................... 63 3.2.3 Reaction Optimisation ....................................................................................... 64 i 3.2.4 Analytical methods............................................................................................ 65 3.3 Results and Discussion ............................................................................................. 71 3.3.1 Soybean 15-lipoxygenase catalysed reaction of DPAn-3 .................................... 71 3.3.2 Optimisation of reaction conditions to favour 7R-dioxygenase activity .............. 73 3.3.3 Product characterisation .................................................................................... 76 3.3.4 Effect of n-alcohol chain length ......................................................................... 80 3.3.5 Application of optimised conditions to other LC-PUFA substrates..................... 82 3.3.6 Analysis of enzyme structure by circular dichrosim spectroscopy ...................... 90 3.3.7 Further discussion ............................................................................................. 94 3.4 Conclusion ............................................................................................................... 98 CHAPTER IV .................................................................................................................... 99 4. Investigating the Anaerobic Lipohydroperoxidase Activity of Soybean 15- Lipoxygenase-1 on LC-PUFA .......................................................................................... 100 4.1 Introduction ............................................................................................................ 100 4.2 Materials and Methods ........................................................................................... 103 4.2.1 Materials ......................................................................................................... 103 4.2.2 Determination of critical micelle concentration................................................ 103 4.2.3 General reaction protocol ................................................................................ 104 4.2.4 Analytical methods.......................................................................................... 104 4.3 Results and Discussion ........................................................................................... 108 4.3.1 Monitoring the metabolism of DHA by 15-sLOX-1 under anaerobic conditions ................................................................................................................ 108 4.3.2 Effect of incubation conditions ........................................................................ 113 ii 4.3.3 Characterisation of anaerobic reaction products ............................................... 118 4.3.4 Analysis of 4-hydroxy-TEMPO radical-scavenging adducts ............................ 128 4.3.5 Use of 2-methoxyphenol as a co-substrate ....................................................... 133 4.3.6 Lipohydroperoxidase activity with other LC-PUFAs ....................................... 134 4.4 Conclusion ............................................................................................................. 138 CHAPTER V ................................................................................................................... 139 5. Covalent Immobilisation of Soybean 15-Lipoxygenase-1 on Various Functionalised Methacrylate Porous Resins to Enable Multiple Reuse of the Enzyme .............................. 140 5.1 Introduction ............................................................................................................ 140 5.2 Materials and Methods ........................................................................................... 145 5.2.1 Materials ......................................................................................................... 145 5.2.2 General immobilisation procedures ................................................................. 145 5.2.3 Bradford protein concentration assay ............................................................... 146 5.2.4 Dissolved oxygen consumption assay .............................................................. 147 5.2.5 Kinetic parameters for soluble (LOXSol) and immobilised LOX (LOXImm) ....... 148 5.2.6 Thermal stability of soluble LOX and immobilised LOX ................................. 148 5.3 Results and Discussion ........................................................................................... 149 5.3.1 Bradford protein determination assay .............................................................. 149 5.3.2 Preliminary screening of immobilisation and optimisation ............................... 150 5.3.3 Optimisation of incubation conditions ............................................................. 153 5.3.4 Application of optimised conditions to complete resin range............................ 163 5.3.5 Estimation of kinetic parameters...................................................................... 166 5.3.6 Effect of immobilisation on pH stability .......................................................... 169 iii 5.3.7 Thermal stability of LOXImm and LOXSol ......................................................... 172 5.3.8 Reusability potential of LOXImm ...................................................................... 176 5.3.9 Analysis of products from linoleic acid catalysed by LOXImm .......................... 178 5.4 Conclusion ............................................................................................................. 180 CHAPTER VI .................................................................................................................. 182 6. Application of Immobilised Soybean 15-Lipoxygenase-1 for the Synthesis of Mono- and Di-Hydroxy Resolvin Analogs ......................................................................................... 183 6.1 Introduction ............................................................................................................ 183 6.2 Materials and Methods ..........................................................................................