Engineering the Substrate Specificity of Galactose Oxidase Lucy Ann Chappell BSc. (Hons) Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Molecular and Cellular Biology September 2013 The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement © 2013 The University of Leeds and Lucy Ann Chappell The right of Lucy Ann Chappell to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. ii Acknowledgements My greatest thanks go to Prof. Michael McPherson for the support, patience, expertise and time he has given me throughout my PhD. Many thanks to Dr Bruce Turnbull for always finding time to help me with the Chemistry side of the project, particularly the NMR work. Also thanks to Dr Arwen Pearson for her support, especially reading and correcting my final thesis. Completion of the laboratory work would not have been possible without the help of numerous lab members and friends who have taught me so much, helped develop my scientific skills and provided much needed emotional support – thank you for your patience and friendship: Dr Thembi Gaule, Dr Sarah Deacon, Dr Christian Tiede, Dr Saskia Bakker, Ms Briony Yorke and Mrs Janice Robottom. Thank you to three students I supervised during my PhD for their friendship and contributions to the project: Mr Ciaran Doherty, Ms Lito Paraskevopoulou and Ms Upasana Mandal. Thanks also to Mr Daniel Williamson for help with the practical side of the NMR work. I am very grateful to many staff members within the Astbury Centre and the Faculty of Biological Sciences for help during my time here, especially Dr Iain Manfield for many fruitful discussions, Dr Gareth Howell for help with imaging software and the ever friendly Stores staff. I greatly acknowledge the financial support from The Wellcome Trust. Finally I offer my deepest gratitude to Dr Kevin Colgan, my future husband, for his love and support, for always being there for me and for keeping me in the real world. iii Abstract Biocatalysis, the use of enzymes to catalyse the generation of specific chemicals, has a number of advantages including high specificity, lower energy requirements and greater sustainability over an equivalent chemical process. Several methods exist for optimisation of enzymes for use in industrial processes, including introduction of mutations to generate libraries of variants which are then screened for the desired properties, such as stability or substrate specificity. Galactose oxidase catalyses the oxidation of primary alcohols to the corresponding aldehyde, with concomitant reduction of dioxygen to hydrogen peroxide. It has already been developed for use in a range of biotechnological processes and is an ideal candidate for further development due to features including high stability, a surface exposed active site displaying broad substrate specificity, and an autocatalytically-generated cofactor. Research presented in this thesis investigates the effect on activity towards a range of alternative substrates of mutations at selected active site residues with the aim of expanding the biotechnological potential of galactose oxidase. Libraries of variants were designed and generated using high quality oligonucleotides constructed using trimer phosphoramidites. Screening assays used by other groups were optimised by varying different components. These assays were then used to identify a number of variants displaying enhanced activity towards D- arabinose, D-glucose, D-xylose or glycerol. A selection of these variants were then further characterised in order to understand the biochemical basis of the altered activities and determine some of the conditions required for potential industrial application of the variants. The most exciting results include identification of a variant displaying higher levels of activity towards glycerol than towards the native substrate D-galactose; determination of the position of oxidation of D-arabinose at the C-4 hydroxyl; and the observation that mutation of Phe194 significantly affects binding of D-glucose in the active site. iv Table of Contents Acknowledgements ................................................................................................................... iii Abstract ..................................................................................................................................... iv Table of Contents ....................................................................................................................... v List of Figures ............................................................................................................................ xi List of Tables ............................................................................................................................ xv Abbreviations ......................................................................................................................... xvii CHAPTER 1 : INTRODUCTION .................................................................. 1 1.1 Exploiting biology for industrial processes ..................................................... 2 1.2 Biocatalysis ................................................................................................... 3 1.3 Optimisation of enzymes for biocatalysis ....................................................... 5 1.3.1 Immobilisation ............................................................................................................. 5 1.3.2 Traditional directed evolution ..................................................................................... 5 1.3.3 The need for smaller libraries ...................................................................................... 6 1.3.4 Reducing the bias problem .......................................................................................... 6 1.3.5 Combining beneficial mutations .................................................................................. 9 1.3.6 Design of mutagenesis strategies .............................................................................. 11 1.3.7 The argument for ‘blind’ evolution ............................................................................ 15 1.3.8 Enzymes in industrial processes ................................................................................ 16 1.4 Screening for altered activities ..................................................................... 17 1.4.1 Selection techniques .................................................................................................. 18 1.4.2 Screening techniques ................................................................................................. 18 1.5 Enzyme cofactors......................................................................................... 21 1.5.1 Metalloenzymes ......................................................................................................... 22 1.6 Galactose oxidase ........................................................................................ 24 1.6.1 Structure of GO .......................................................................................................... 26 1.6.2 Substrate specificity & binding .................................................................................. 31 1.6.3 Mechanism of alcohol oxidation ................................................................................ 35 1.6.4 Expression and purification of recombinant GO........................................................ 39 v 1.6.5 Uses of GO .................................................................................................................. 40 1.7 Aims of the project...................................................................................... 44 CHAPTER 2 : MATERIALS & METHODS ................................................... 47 2.1 Growth media ............................................................................................. 48 2.1.1 2TY broth .................................................................................................................... 48 2.1.2 2TY agar plates ........................................................................................................... 48 2.1.3 ZYP-0.8G broth ........................................................................................................... 48 2.1.4 8ZYM+5052+25 mM succinate broth ......................................................................... 48 2.1.5 BMMY broth ............................................................................................................... 49 2.1.6 SOC broth ................................................................................................................... 49 2.1.7 Antibiotics ................................................................................................................... 49 2.2 E. coli .......................................................................................................... 49 2.2.1 XL1 blue ...................................................................................................................... 49 2.2.2 BL21 Star (DE3) ..........................................................................................................