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The Heterogeneity of Amine Oxidase within Rhodococcus opacus Alexander Foster Submitted for the degree of Doctor of Philosophy Heriot-Watt University School of Engineering and Physical Sciences Department of Chemical Engineering March 2013 The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information. Abstract Four native amine oxidases have been identified from Rhodococcus opacus to reveal phenotypic plasticity and catalytic activity with respect to structurally diverse natural and synthetic amines. Altering the amine growth substrate enabled tailored and targeted oxidase upregulation, facilitating catalytic differentiation and isolation. Each enzyme was purified over 80 fold by chromatography, allowing subsequent characterisation. Two oxidases possessed a copper dependent redox co-factor with broad specificity towards monoamines. Michaelis constants (KM) ranged from 0.1 to 0.9 mM for common C1–C5 aliphatic monoamines and <0.2 mM for a range of aromatic amines. The remaining two oxidases by contrast were highly specific for aliphatic diamines, with a Michaelis constants (KM) = 60 µM for putrescine by a third copper oxidase and a (KM) = 190 µM by a flavin dependent oxidase. MALDI-TOF and genomic analysis has indicated metabolic gene clusters, multiple gene activation, and complex biodegradation pathways. With a consideration of the diamine acting oxidase, a putrescine degradation pathway is confirmed that utilises oxidases in tandem with a 4-aminobutyraldehyde dehydrogenase. The taxonomic distribution of this pathway is further examined utilising phylogenetic analysis. Oxidase regulation and integration into the nitrogen cycle is then considered, with implications in bioremediation and biocatalysis discussed. i Dedication I would like to dedicate my thesis to my family, who for years have given me belief, encouragement and support. My wonderful wife Melissa who I love and adore, my daughter Samantha whose smile and laughter is the best inspiration a dad could have, and also “Bones” who sadly passed away a few months ago. ii Acknowledgements First and foremost I would like to thank Mark Keane. Over the last 5 years he has given me an immense amount of support, pushing me to achieve my very best, offering considerable direction, encouragement and patience. He is a very good supervisor and became a very good friend. I would also like to acknowledge the assistance of Peter Morris, and the students from within his group, particularly Callum Scott, Angela Bell and Charlotte Wendelboe-Nelson. Much of my research was dependent upon working within his laboratory, in addition to their help I gained numerous ideas and different scientific perspectives. Finally I would like to thank Robert Speight and Nicole Barnes of Ingenza, my industrial supervisors who provided the initial scientific support, and subsequently helped the publication of several papers. iii ACADEMIC REGISTRY Research Thesis Submission Name: Alexander Brett Foster School/PGI: School of Engineering and Physical Sciences - Chemical Engineering Version: (i.e. First, First Degree Sought Doctor of Philosophy – Chemical Resubmission, Final) (Award and Engineering Subject area) Declaration In accordance with the appropriate regulations I hereby submit my thesis and I declare that: 1) the thesis embodies the results of my own work and has been composed by myself 2) where appropriate, I have made acknowledgement of the work of others and have made reference to work carried out in collaboration with other persons 3) the thesis is the correct version of the thesis for submission and is the same version as any electronic versions submitted*. 4) my thesis for the award referred to, deposited in the Heriot-Watt University Library, should be made available for loan or photocopying and be available via the Institutional Repository, subject to such conditions as the Librarian may require 5) I understand that as a student of the University I am required to abide by the Regulations of the University and to conform to its discipline. * Please note that it is the responsibility of the candidate to ensure that the correct version of the thesis is submitted. Signature of Date: Candidate: Submission Submitted By (name in capitals): Signature of Individual Submitting: Date Submitted: For Completion in the Student Service Centre (SSC) Received in the SSC by (name in capitals): 1.1 Method of Submission (Handed in to SSC; posted through internal/external mail): 1.2 E-thesis Submitted (mandatory for final theses) Signature: Date: iv Table of Contents Abstract .............................................................................................................................. i Dedication ......................................................................................................................... ii Acknowledgements .......................................................................................................... iii Table of Contents .............................................................................................................. v List of Figures ................................................................................................................ viii List of Tables................................................................................................................... xii List of Publications by Candidate .................................................................................. xiv Glossary .......................................................................................................................... xv Scope and Organisation of the Thesis ............................................................................ xvi Chapter 1 Identification, Functional Expression and Kinetic Analysis of Two Primary Amines Oxidases from Rhodococcus opacus 1.1 Introduction ........................................................................................................ 1 1.2 Materials and Methods. ...................................................................................... 4 1.2.1 Bacterial strain and culture conditions ............................................................ 4 1.2.2 Purification of amine oxidase ......................................................................... 5 1.2.3 AO Gel activity staining ................................................................................. 5 1.2.4 In-gel digestion of protein spots and protein identification ............................ 6 1.2.5 Enzyme assay .................................................................................................. 7 1.2.6 Enzyme inhibition ........................................................................................... 7 1.2.7 UV spectra analysis ........................................................................................ 8 1.3 Results and Discussion ....................................................................................... 8 1.3.1 Amine oxidase induction ................................................................................ 8 1.3.2 Purification of oxidases post induction ......................................................... 12 1.3.3 Enzyme characterisation ............................................................................... 18 1.3.4 Proteomic identification and genomic analysis ............................................ 21 1.3.5 Kinetic Analysis ............................................................................................ 23 1.3.6 Analysis of AO1 kinetics .............................................................................. 26 1.3.7 Analysis of AO2 kinetics .............................................................................. 30 1.4 Conclusions ...................................................................................................... 31 1.5 References ........................................................................................................ 32 Chapter 2 - Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus 2.1 Introduction ...................................................................................................... 37 2.2 Materials and methods ...................................................................................... 39 2.2.1 Bacterial strain and culture conditions .......................................................... 39 2.2.2 Purification of amine oxidase ....................................................................... 40 2.2.3 In-gel digestion of protein spots and protein identification .......................... 41 2.2.4 Peroxide assay............................................................................................... 42 2.2.5 Enzyme inhibition ......................................................................................... 42 2.2.6 UV spectra analysis ...................................................................................... 43 2.3 Results and discussion ...................................................................................... 43 2.3.1 Regulation of diamine oxidase expression ................................................... 43 v 2.3.2 Inhibitor specificity ....................................................................................... 47 2.3.3 Oxidase purification and isolation ...............................................................

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