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Phd Thesis.Pdf Investigating the industrial application potentials of cytochrome P450 BM-3 and four novel putative cytochrome P450s isolated from Cupriavidus necator H16 By: Inas Al-nuaemi A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Engineering Department of Chemical and Biological Engineering Jun 2018 Dedicated to the memory of my husband, Mohammed, who always believed in my ability to succeed in the academic field. You are gone but your belief in me has made this journey possible Acknowledgements I would like to thank The University of Sheffield, Ministry of Higher Education in Iraq, Iraqi Cultural Attache in London, The Middle Technical University in Iraq and the Institute of Technology in Baghdad, Iraq for offering me this valuable experience and for their financial support. I would like to express my immense gratitude to my supervisor Dr Tuck Wong for his unconditional patience and guidance throughout the entire time of my PhD. It would have been a very different story without his inspiring and motivating discussions filled with knowledge and expertise. I would like to show my appreciation to Dr Kang Lan Tee whose constant support, positive attitude and invaluable advice has made the project to proceed and succeed! Everyone with Tuck’s group Abdulrahman, Miriam, Jose, Yomi, Pawel, Zaki and Hossam I would like to thank you for all your amazing help and support, I have been very fortunate to share lab space with you all. Lastly, I would like to thank my family to whom I owe a great deal. I would like to thank my father Jabbar and my mother Rajaa and my sisters Wasan. Usra, Zahraa and Huda and my sister in law Masara who believed I could do this even in the hardest of times and stood by me with interest, enthusiasm and a completely unrealistic sense of pride in me. Also many thanks to my friend Marwa and her husband Mohammed for your outstanding, continues support. And finally, the one person who has made this all possible has been my son Abdullah. He has been a constant source of support and encouragement for me to continue my PhD. Hence, great appreciation and enormous thanks are due to him, for without his understanding, I am sure this thesis would never have been completed. Declaration The author declares that no portion of the work referred to in this thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. II Contents page I. List of abbreviations ………………………………………………….…….VIII II. List of figures …………………………………………………………..….….XI III. List of tables ……………………………………………………………… XVII 1 Introduction ................................................................................................................... 1 1.1 Objectives.............................................................................................................................1 1.1.1 Part II: Cytochrome P450 BM-3 ...................................................................................2 1.1.2 Part III: Putative cytochrome P450s from Cupriavidus necator H16 ...........................3 1.2 Organisation of this thesis ...................................................................................................4 2 Heme proteins ............................................................................................................... 7 2.1 Heme structure & function ..................................................................................................7 2.2 Structural analysis of heme proteins ...................................................................................8 2.3 Heme enzymes .................................................................................................................. 10 2.3.1 Cytochrome P450s .................................................................................................... 11 2.3.2 Nitric oxide synthases ............................................................................................... 12 2.3.3 Peroxidases ............................................................................................................... 13 2.3.4 Chloroperoxidases .................................................................................................... 14 2.3.5 Heme oxygenases ..................................................................................................... 15 2.3.6 Dioxygenases ............................................................................................................. 16 2.3.7 Diheme proteins........................................................................................................ 17 3 Cytochrome P450 superfamily .................................................................................. 19 3.1 Discovery ........................................................................................................................... 19 3.2 Nomenclature ................................................................................................................... 19 3.3 Evolution ........................................................................................................................... 20 3.4 Structure ........................................................................................................................... 22 3.5 Biochemistry ..................................................................................................................... 25 3.5.1 Optical properties ..................................................................................................... 25 III 3.5.2 Catalytic cycle ............................................................................................................ 27 3.6 Cytochrome P450-catalysed reactions ............................................................................. 29 3.6.1 Hydroxylation ............................................................................................................ 30 3.6.2 Epoxidation ............................................................................................................... 31 3.6.3 Oxidation of aromatic rings....................................................................................... 31 3.6.4 Dealkylation .............................................................................................................. 31 3.6.5 Unusual P450 reactions ............................................................................................ 32 3.7 Industrial applications of cytochrome P450s .................................................................... 34 4 Introduction to cytochrome P450 BM-3 ................................................................... 39 4.1 Domain architecture ......................................................................................................... 39 4.2 Protein expression ............................................................................................................ 40 4.3 Protein structure ............................................................................................................... 41 4.4 Dimerisation ...................................................................................................................... 44 4.5 Electron transfer and redox partners ............................................................................... 45 4.6 Protein engineering .......................................................................................................... 46 4.7 Immobilisation .................................................................................................................. 51 4.8 Industrial applications of cytochrome P450 BM-3............................................................ 53 5 Biocatalysis in non-conventional solvents ................................................................. 55 5.1 Tolerance vs stability ......................................................................................................... 55 5.2 Solvent classification ......................................................................................................... 56 5.3 Biocatalysis in organic media ............................................................................................ 59 5.3.1 Neat solvent .............................................................................................................. 59 5.3.2 Water-solvent mixture (co-solvent) .......................................................................... 61 5.3.3 Biphasic system ......................................................................................................... 62 5.4 Biocatalysis in micellar systems ........................................................................................ 64 5.5 Biocatalysis in ionic liquids ................................................................................................ 65 5.6 Biocatalysis in supercritical fluids ..................................................................................... 67 5.7 Stabilisation of enzymes in non-conventional solvents .................................................... 68 5.8 Tolerance of cytochrome P450 BM-3 to organic co-solvents ........................................... 73 5.8.1 Mutants of F87A parent and their tolerance to organic co-solvents ....................... 78 6 Materials and Methods ..............................................................................................
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