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Exploitation of the Potential of a Novel Bacterial Peroxidase for the Development of a New Biocatalytic Process

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Exploitation of the Potential of a Novel Bacterial Peroxidase for the Development of a New Biocatalytic Process EXPLOITATION OF THE POTENTIAL OF A NOVEL BACTERIAL PEROXIDASE FOR THE DEVELOPMENT OF A NEW BIOCATALYTIC PROCESS By AMOS MUSENGI Thesis submitted in partial fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness Sciences At the Cape Peninsula University of Technology Supervisor: Prof. Stephanie Burton Co-supervisors: Prof. Brett Pletschke, Dr Marilize Le Roes-Hill Bellville February 2014 DECLARATION I, AMOS MUSENGI, declare that the contents of this dissertation/thesis represent my own unaided work, and that the thesis has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the Cape Peninsula University of Technology. 14 February 2014 Signed Date ii ABSTRACT Peroxidases are ubiquitous catalysts that oxidise a wide variety of organic and inorganic compounds employing peroxide as the electron acceptor. They are an important class of oxidative enzymes which are found in nature, where they perform diverse physiological functions. Apart from the white rot fungi, actinomycetes are the only other known source of extracellular peroxidases. In this study, the production of extracellular peroxidase in wild type actinomycete strains was investigated, for the purpose of large-scale production and finding suitable applications. The adjustment of environmental parameters (medium components, pH, temperature and inducers) to optimise extracellular peroxidase production in five different strains was carried out. Five Streptomyces strains isolated from various natural habitats were initially selected for optimisation of their peroxidase production. Streptomyces sp. strain BSII#1 and Streptomyces sp. strain GSIII#1 exhibited the highest peroxidase activities (1.30±0.04 U ml-1 and 0.757±0.01 U ml-1, respectively) in a complex production medium at 37°C and pH 8.0 in both cases. Maximum enzyme production for Streptomyces strain BSII#1 was obtained in the presence of 0.1 mM veratryl alcohol or pyrogallol, while 0.1 mM guaiacol induced the highest peroxidase production in Streptomyces sp. strain GSIII#1. As the highest peroxidase producer, Streptomyces sp. strain BSII#1 was selected for further studies. The strain was first characterised by a polyphasic approach, and was shown to belong to the genus Streptomyces using various chemotaxonomic, genotypic and phenotypic tests. Production of peroxidase was scaled up to larger volumes in different bioreactor formats. The airlift configuration was optimal for peroxidase production, with Streptomyces sp. strain BSII#1 achieving maximum production (4.76±0.46 U ml-1) in the 3 l culture volume within 60 hrs of incubation. A protocol for the purification of the peroxidase was developed, which involved sequential steps of acid and acetone precipitation, as well as ultrafiltration. A purification factor of at least 46-fold was achieved using this method and the protein was further analysed by LC- MS. The protein was shown to be a 46 kDa protein, and further biochemical characterisation showed that the peroxidase had a narrower spectrum of substrates as compared to reports on other peroxidases derived from actinomycetes. With 2,4-dichlorophenol as the substrate, -1 the Km and Vmax for this enzyme were 0.893 mM and 1.081 µmol min , respectively. The purified peroxidase was also capable of catalysing coupling reactions between several phenolic monomer pairs. iii Overall, the peroxidase from Streptomyces sp. strain BSII#1 could feasibly be produced in larger scales and there remains further room to investigate other potential applications for this enzyme. iv ACKNOWLEDGEMENTS . I am grateful to my supervisor, Professor Stephanie Burton, for giving me the privileged opportunity to grow as a scientist and as an individual. I acknowledge my co-supervisor, Professor Pletschke, for all the helpful guidance, encouragement and mentorship. I am indebted to Dr Marilize Le Roes-Hill for being the capable leader she is, for carefully following all my work through and always believing in me, even at my weakest times and in all my short-comings, but encouraging me all the way. I express my heartfelt gratitude to Dr Nuraan Khan for teaching me the ropes inside and outside the laboratory, and for going out of her way to working lengthy, extra hours to ensure the success of my work. I have benefited tremendously from your close mentorship. My profound thanks go to Dr Tukayi Kudanga for being the mentor he has been to me, both academically and on life’s growing up issues. Special thanks go to Mr Alaric Prins for being extra handy and helpful at all times; from calibrating/operating laboratory devices to helping format the thesis, your all- round help has just been awesome and really uplifting - I would not have made it without your handy hints and tips. I am most grateful to Miss Kim Durrell for being the constant friend she has been to me both in and out of season - through the hard times you have been a pillar of strength and your encouragement kept me going when things didn’t seem to be working. Your kind assistance with the experiments in the lab is much appreciated. I am also greatly indebted to the entire BTB family for their love and constant support - the administrators, the post-docs and fellow students, thank you all. Special thanks go to Professor Syldatk and his research group at the KIT (Karlsruhe Institute of Technology) Institut für Bio- und Lebensmitteltechnik Bereich II: Technische Biologie for all that I learned on their fermentation systems. Special thanks extended to my mentors at the Harare Institute of Technology, Dr Perkins Muredzi and Mr Dinga N. Moyo for always supporting all my academic endeavours. I greatly appreciate my entire family for always supporting me, and my fiancée for her patience through the long period of physical absence. Your prayers and love have pulled me through the dark and rough patches, thank you for encouraging and standing by me. And to the Almighty God who makes things good in His perfect timing, be all the glory and honour forevermore. v . The financial assistance of the National Research Foundation towards this research is acknowledged. Opinions expressed in this thesis and the conclusions arrived at, are those of the author, and are not necessarily to be attributed to the National Research Foundation. Assistance of the Water Research Commission of South Africa for bursary funding is also greatly acknowledged. vi DEDICATION Molly Musengi Gone too soon, beloved sister, wonderful friend Your life has been short, way too short, but was an example well lived. Rest in eternal peace. Love you always vii LIST OF PUBLICATIONS The following is a list of the publication and conference presentations resulting from the work presented in this thesis. Publication Musengi, A., Khan, N., Le Roes-Hill, M., Pletschke, B.I., and Burton, S.G., 2013. Increasing the scale of peroxidase production by Streptomyces sp. strain BSII#1. Journal of Applied Microbiology, in press; doi:10.1111/jam.12380. Conference Papers 1. CPUT research day, November 2011, Cape Town Campus. Production of peroxidase from selected actinomycete strains. A Musengi, N Khan, M Le Roes-Hill, B Pletschke, D Cowan, S Burton (poster presentation). 2. 16th International Symposium on the Biology of Actinomycetes, December 2011 (Puerto Vallerta, Mexico). In search of novel peroxidases from Actinobacteria. N Khan, A Musengi, K Durrell, M Le Roes-Hill, D Cowan, S Burton (poster presentation). 3. South African Society of Biochemistry and Molecular Biology, Federation of African Societies of Biochemistry and Molecular Biology, January 2012 (Drakensberg, South Africa).Production of peroxidase from selected actinobacterial strains. A Musengi, N Khan, M Le Roes-Hill, BI Pletschke, DA Cowan, SG Burton (poster presentation). 4. Joint conference on Science and Technology for development in Africa, Cape Town, June 2012. Production of peroxidase from selected actinomycete strains. A Musengi, N Khan, M Le Roes-Hill, BI Pletschke, DA Cowan and SG Burton (oral presentation). 5. CPUT research day, November 2012, Cape Town Campus. Purification of peroxidase from a novel actinobacterial strain. A Musengi, N Khan, M Le Roes-Hill, B Pletschke, D Cowan, SG Burton (Third prize for Best Research Poster presentation). 6. South African Society of Microbiology, November 2013, Bela-Bela Hot Springs, Limpopo Province. Increasing the scale of peroxidase production by Streptomyces sp. strain BSII#1. A Musengi, N Khan, M Le Roes-Hill, B Pletschke, SG Burton. viii TABLE OF CONTENTS Contents DECLARATION .......................................................................................................... II ABSTRACT ................................................................................................................ III ACKNOWLEDGEMENTS .......................................................................................... V DEDICATION ........................................................................................................... VII LIST OF PUBLICATIONS ....................................................................................... VIII TABLE OF CONTENTS ............................................................................................ IX LIST OF FIGURES ................................................................................................... XV LIST OF TABLES ..................................................................................................
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