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Production and Molecular Characterization of Peroxidases

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Production and Molecular Characterization of Peroxidases PRODUCTION AND MOLECULAR CHARACTERIZATION OF PEROXIDASES FROM NOVEL LIGNINOLYTIC PROTEOBACTERIA AND BACILLUS STRAINS FALADE AYODEJI OSMUND 2018 PRODUCTION AND MOLECULAR CHARACTERIZATION OF PEROXIDASES FROM NOVEL LIGNINOLYTIC PROTEOBACTERIA AND BACILLUS STRAINS BY FALADE AYODEJI OSMUND (201508654) A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY FACULTY OF SCIENCE AND AGRICULTURE UNIVERSITY OF FORT HARE ALICE, 5700 SOUTH AFRICA SUPERVISOR: PROF. L. V. MABINYA CO-SUPERVISOR: PROF. U. U. NWODO 2018 DECLARATION I, the undersigned, declare that this thesis entitled “Production and molecular characterization of peroxidases from novel ligninolytic proteobacteria and bacillus strains” submitted to the University of Fort Hare for the degree of Doctor of Philosophy in Biochemistry in the Faculty of Science and Agriculture is my original work and that the work has not been submitted to any other University in partial or entirely for the award of any degree or examination purposes. Name: Falade Ayodeji Osmund Signature: ………………………… Date: ………………………… ii DECLARATION ON PLAGIARISM I, Falade Ayodeji Osmund, student number: 201508654 hereby declare that I am fully aware of the University of Fort Hare’s policy on plagiarism and I have taken every precaution to comply with the regulations. Signature.………………………….. Date.………………………….. iii DECLARATION ON RESEARCH ETHICAL CLEARANCE I, Falade Ayodeji Osmund, student number: 201508654 hereby declare that I am fully aware of the University of Fort Hare’s policy on research ethics and I have taken every precaution to comply with the regulations. I confirm that my research constitutes an exemption to Rule G17.6.10.5 and an ethical certificate with a reference number is not required. Signature.………………………….. Date.………………………….. iv CERTIFICATION This thesis entitled “Production and molecular characterization of peroxidases from novel ligninolytic proteobacteria and bacillus strains” meets the regulation governing the award of degree of Doctor of Philosophy of the University of Fort Hare and is approved for its contribution to scientific knowledge. ……………………………………… …………………………… Prof. L. V. Mabinya Date Supervisor …………………………………… …………………………… Prof. U. U. Nwodo Date Co-supervisor and Head of Department v DEDICATION This thesis is dedicated to God Almighty, the giver of life; and in loving memory of my late Father, Mr. Johnson Akin Falade, who passed on in the course of this study. “…it is not of him that willeth, nor of him that runneth, but of God that sheweth mercy” Rom. 9:16 (KJV) vi ACKNOWLEDGMENTS Firstly, I humbly and sincerely thank the Almighty God for the gift of life and for blessing me with the required wisdom for this study as “…a man can receive nothing, except it be given him from heaven”. I wish to express my profound gratitude to Prof. A. I. Okoh for accepting me for a PhD study in his prestigious research group, Applied and Environmental Microbiology Research Group (AEMREG). No doubt, his kind gesture has in no small measure contributed to the realization of my academic dream. I would like to express my heartfelt appreciation to my supervisors, Prof. L. V. Mabinya and Prof. U. U. Nwodo for their constructive criticisms, inputs, corrections and insightful comments. Beyond their supervisory roles, they have been sources of inspiration to me throughtout the period of this study. Their wealth of experience has in no doubt contributed immensely to the success of this study. The technical supports and encouragements from my senior colleague, Dr. (Mrs) Evlyn Fatokun and the following colleagues: John Unuofin, Charles Osunla, Taiwo Fadare, Tayo Abioye, Kayode Afolabi, Tennison Digban, Rev. Sister Eyisi, Nonso Nnolim, Olusesan Adelabu, Tosin Olasehinde, Cyrus Ekundayo and Aboi Igwaran are gratefully acknowledged. The advice and supports of the following post doctoral fellows: Dr. Ben Iweriebor, Dr. Kunle Okayeito, Dr. Martins Adefisoye, Dr. Nolwazi Bhembe and Dr. Goke Adeniji are also acknowledged. More so, I wish to extend my sincere appreciation to all members of AEMREG and Biocatalysis/Biomolecules sub-group for their constructive criticisms and contributions toward the success of this study. Furthermore, I am very grateful to my mother, Mrs. Victoria Ajibola Falade and my parents-in- law, Chief and Mrs. Ayodele Omotayo for their prayers, encouragements and financial supports throughout the period of this study. May the good Lord bless you with long lives to enjoy the fruit of your labour in Jesus name (Amen). I would like to specially thank my sister, Mrs. Funmilayo Olaitan for her financial contribution towards the success of my PhD study, you have been a pillar of support; which has always proven to me that “blood is thicker than water”. May God reward your labour of love in Jesus name (Amen). Similarly, the moral and financial supports from my brother, Mr. Kole Falade and brothers-in-law, Messrs Ope, Seun, Lekan and Tubosun Omotayo vii are gratefully acknowledged. I would also like to thank my brother, Mr. Israel Orimoloye for his moral support during this study. This study would not have been a success without the prayers and support of my beautiful jewel, Mrs. Monisola Ayodeji-Falade, thank you for believing in my dreams and aspirations. I love you. This section would not be completed without appreciating my lovely kids: Praise and Caleb for their understanding and endurance throughout the period of this study. Daddy loves you. I thank Dr. J. O. Obameso for designing the primers used in this study. I also thank Dr. A. B. Falowo for proofreading part of this thesis and for his moral support. More so, I thank the management of the Federal University of Technology, Akure, Nigeria for granting me leave of absence to pursue my PhD programme. In conclusion, the financial supports from the National Research Foundation (NRF) under the South Africa/Tunisia bilateral collaboration (grant number: 95364), the South African Medical Research Council (SAMRC) and the Govan Mbeki Research and Development Centre (GMRDC) of the University of Fort Hare are gratefully acknowledged. viii LIST OF PUBLICATIONS FROM THIS THESIS Journal articles i. Falade, A. O., Nwodo, U. U., Iweriebor, B. C., Green, E., Mabinya, L. V. and Okoh, A. I. (2017). Lignin peroxidase functionalities and prospective applications. Microbiology Open 6, e00394. ii. Falade, A. O., Eyisi, O. A. L., Mabinya, L. V., Nwodo, U. U. and Okoh, A. I. (2017). Peroxidase production and ligninolytic potentials of freshwater bacteria Raoultella ornithinolytica and Ensifer adhaerens. Biotechnology Reports 16, 12-17. Conference proceedings i. Falade, A. O., Mabinya, L. V., Jaouani, A., Nwodo, U. U. and Okoh, A. I. (2016). Ligninolytic and peroxidase production potentials of novel bacterial strains isolated from freshwater and terrestrial milieux of the Nkonkobe Municipality, Eastern Cape, South Africa. Proceedings of the 1st Tunisian-South African International Conference, ISBST, Sidi Thabet, Tunisia (4 – 6 November, 2016). Oral. ii. Falade, A. O., Mabinya, L. V., Nwodo, U. U. and Okoh, A. I. (2018). Peroxidase production by Raoultella ornithinolytica OKOH-1 isolated from Tyhume river sediments. Proceedings of the Biennial Conference of the South African Society for Microbiology (SASM) held in Misty Hills Hotel and Conference Centre, Muldersdrift, Johannesburg, South Africa (4 – 7 April, 2018). Oral. ix LIST OF SUBMITTED MANUSCRIPTS i. Versatile peroxidase functionality: elimination of endocrine disrupting chemicals in wastewater. ii. Peroxidase produced by ligninolytic Bacillus species isolated from marsh and grassland decolourized anthraquinone and azo dyes. iii. Optimized peroxidase production and detection of catalase-peroxidase gene (KatG) in a Bacillus species isolated from Hogsback forest reserve, South Africa. iv. Optimization of process parameters for exoperoxidase production by Ensifer adhaerens NWODO-2 and PCR detection of catalase-peroxidase gene (KatG). v. Peroxidase production by a novel ligninolytic proteobacteria strain: Raoultella ornithinolytica OKOH-1. vi. Biochemical and molecular characterization of Raoultella ornithinolytica peroxidase with biotechnological potentials in dye decolourization and development of cosmetic agent. x LIST OF ACRONYMS 2, 4-DCP: 2, 4-Dichlorophenol ABTS: 2,2´-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) AC: Ammonium Chloride AEMREG: Applied and Environmental Microbiology Research Group AN: Ammonium Nitrate ANOVA: Analysis of Variance APEs: Alkylphenol Ethoxylates APs: Alkylphenols AS: Ammonium Sulphate AZB: Azure B B[a]P: Benzo [a] Pyrene BBD: Box-Behnken Design BGL: β-glucosidase BLAST: Basic Local Alignment Search Tools BPA: Bisphenol A BSA: Bovine Serum Albumin CCD: Central Composite Design CcP: Cytochrome c Peroxidase CLEA: Cross Linked Enzyme Aggregates CR: Congo Red DDT: Dichlorodiphenyltrichloroethane DiHCcP: Di-heme Cytochrome c Peroxidase DNA: Deoxyribonucleic Acid DOPA: Dihydroxyphenylalanine DyP: Dye decolourizing Peroxidase EDC: Endocrine Disrupting Chemicals EDTA: Ethylenediaminetetraacetic acid EMR: Enzymatic Membrane Reactor FPase: Filter Paper Cellulase GA: Guaiacol xi GMRDC: Govan Mbeki Research and Development Centre HPI: Hydroperoxidase I HRP: Horseradish Peroxidase KatG: Catalase-peroxidase KL: Kraft Lignin KTBA: α-keto-γ-thiomethylbutyric acid LiP: Lignin Peroxidase LMEs: Lignin Modifying Enzymes LRET: Long Range Electron Transfer MEGA: Molecular Evolutionary Genetics Analysis MCO: Multicopper
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