Protein Engineering of a Dye Decolorizing Peroxidase from Pleurotus ostreatus For Efficient Lignocellulose Degradation Abdulrahman Hirab Ali Alessa 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 September 2018 ACKNOWLEDGEMENTS Firstly, I would like to express my profound gratitude to my parents, my wife, my sisters and brothers, for their continuous support and their unconditional love, without whom this would not be achieved. My thanks go to Tabuk University for sponsoring my PhD project. I would like to express my profound gratitude to Dr Wong for giving me the chance to undertake and complete my PhD project in his lab. Thank you for the continuous support and guidance throughout the past four years. I would also like to thank Dr Tee for invaluable scientific discussions and technical advices. Special thanks go to the former and current students in Wong’s research group without whom these four years would not be so special and exciting, Dr Pawel; Dr Hossam; Dr Zaki; Dr David Gonzales; Dr Inas,; Dr Yomi, Dr Miriam; Jose; Valeriane, Melvin, and Robert. ii SUMMARY Dye decolorizing peroxidases (DyPs) have received extensive attention due to their biotechnological importance and potential use in the biological treatment of lignocellulosic biomass. DyPs are haem-containing peroxidases which utilize hydrogen peroxide (H2O2) to catalyse the oxidation of a wide range of substrates. Similar to naturally occurring peroxidases, DyPs are not optimized for industrial utilization owing to their inactivation induced by excess amounts of H2O2. Furthermore, DyPs are active only under acidic conditions and typically lose activity at neutral or alkaline pH. A dye decolorizing peroxidase from the Pleurotus ostreatus (Pleos-DyP4) was identified recently as a first fungal DyP oxidizing Mn2+ to Mn3+ similar to other fungal peroxidases. However, despite its unique pH and thermal stability, similar to other DyPs, it is not suited for industrial applications. Protein engineering methods are widely used to enhance the stability and catalytic efficiency of biocatalysts to render them suitable for industrial purposes. Different directed evolution approaches (namely, error-prone PCR and saturation mutagenesis) were used to construct mutant libraries of DyP4. For protein expression studies, the mutant enzymes were co-expressed with OsmY protein (a novel secretion-enhancing protein) in order to secrete intracellular protein into the media and hence facilitate the screening of mutants. ABTS assay was used to screen for mutants with improved activities in 96-well microtiter plates. Four rounds of error-prone PCR (epPCR) and saturation mutagenesis led to the identification of a mutant with an approximately 10-fold improvement in total activity and resistance to H2O2 inactivation in comparison with the wild type (WT). This study showcases the usefulness of the OsmY-based secretion mechanism of protein in E. coli as a tool in facilitating the screening of DyP4 mutants, and potentially of other heterologous protein variants in E. coli – the preferred host for expression and directed evolution studies. iii Chapter (6) is written for a manuscript and will be submitted for publication. Abdulrahman HA Alessa, David Gonzalez Perez, Hossam EM Omar Ali, Alex Trevaskis, Kang Lan Tee, Xu, Jun and Tuck Seng Wong. 2019. Engineering of DyP4 using an OsmY- based secretion mechanism to facilitate the directed evolution approach in bacteria. Bioresources and Bioprocessing. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................. ii SUMMARY ....................................................................................................................... iii TABLE OF CONTENTS ..................................................................................................... v LIST OF FIGURES ............................................................................................................. x LIST OF TABLES ............................................................................................................ xvi Appendix ........................................................................................................................ xviii Abbreviation ..................................................................................................................... xix CHAPTER 1 Introduction and literature review .............................................................. 1 1.1 Introduction ........................................................................................................... 2 1.2 Lignocellulose as a source of energy .................................................................... 3 1.2.1 Lignocellulose composition ........................................................................... 3 1.2.2 Pretreatment of lignocellulose ....................................................................... 8 1.2.3 White-rot and brown-rot fungi ..................................................................... 13 1.3 Lignocellulose-degrading enzymes ..................................................................... 13 1.3.1 Laccases ....................................................................................................... 14 1.3.2 Peroxidases .................................................................................................. 14 1.4 Potential application of peroxidases .................................................................... 26 1.4.1 Bioremediation ............................................................................................. 26 1.4.2 Odour pollution ............................................................................................ 29 1.4.3 Bioremediation of azo dyes ......................................................................... 29 1.4.4 Limitations of DyP4 for industrial-scale utilization .................................... 30 1.5 Protein engineering ............................................................................................. 32 1.5.1 Directed evolution ........................................................................................ 32 1.5.2 Rational design............................................................................................. 35 1.5.3 Screening...................................................................................................... 36 CHAPTER 2 Materials and methods ............................................................................. 39 2.1 Materials .............................................................................................................. 40 2.2 Media preparation ............................................................................................... 41 2.2.1 2× TY media ................................................................................................ 41 2.2.2 TY AIM media ............................................................................................. 41 2.2.3 Tryptone yeast extract agar plates................................................................ 41 v 2.3 Molecular cloning methods ................................................................................. 41 2.3.1 DNA plasmid isolation ................................................................................ 41 2.3.2 DNA gel extraction (PCR purification) ....................................................... 42 2.3.3 PCR clean-up ............................................................................................... 43 2.3.4 DNA gel electrophoresis .............................................................................. 43 2.4 Bacterial transformation methods ....................................................................... 44 2.4.1 Calcium chloride method ............................................................................. 44 2.4.2 Electroporation method ................................................................................ 45 2.5 Molecular cloning of DyP4 ................................................................................. 46 2.5.1 Cloning of DyP4-tag into pET24a ............................................................... 46 2.5.2 Cloning of pET24a-DyP4-without tag ......................................................... 46 2.6 Protein expression ............................................................................................... 47 2.6.1 Expression of pET24a-DyP4 ....................................................................... 47 2.6.2 Optimization of DyP4 secretion using a pET24a-OsmY-DyP4 vector ....... 48 2.7 Analysis of protein expression ............................................................................ 48 2.7.1 SDS-PAGE .................................................................................................. 48 2.7.2 Preparation of protein samples..................................................................... 49 2.7.3 Activity assays for DyP4 ............................................................................. 50 2.8 Purification .......................................................................................................... 51 2.8.1 Ion-exchange chromatography..................................................................... 51 CHAPTER 3 Encapsulation of DyP4 into the encapsulin nanocompartment