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Xuejun Yu Dissertation Final UNIVERSITY OF CALIFORNIA RIVERSIDE Conversion of Carbon Dioxide to Formate by a Formate Dehydrogenase from Cupriavidus necator A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Bioengineering by Xuejun Yu September 2018 Dissertation Committee: Dr. Ashok Mulchandani, Co-Chairperson Dr. Xin Ge, Co-Chairperson Dr. Russ Hille Copyright by Xuejun Yu 2018 The Dissertation of Xuejun Yu is approved: Committee Co-Chairperson Committee Co-Chairperson University of California, Riverside ACKNOWLEDGEMENTS I would like to express sincere appreciation to my advisor, Professor Ashok Mulchandani, for accepting me to be his student when I was suffering. Thank you very much for your delicate guidance, support and encouragement during the past years. You not only guide me on my PhD study, but also help me to be mature on my personality. From bottom of my heart, I feel very lucky to have you as professor. Also, many thanks go to my other committee members. Professor Xin Ge acted as my co-advisor and provided many valuable comments on my molecular cloning work. Professor Russ Hille has also provided insights, encouragements and advices as a member of my committee members. I have learned so many important insights from our meetings and discussions and thank you for bringing me into the molybdenum/tungsten enzyme conference. I am also thankful to the past and current group members, colleagues and friends - Dimitri Niks, Pankaj Ramnani, Feng Tan, Rabeay Hassan, Trupti Terse, Thien-Toan Tran, Claudia Chaves, Jia-wei Tay, Hui Wang, Pham Tung, Hilda Chan, Yingning Gao and Tynan Young. I had a great time working with you all, and I always feel lucky to know these wonderful people. Your friendship and support will be a wonderful memory to my PhD life. I would especially like to acknowledge the tremendous support from Dimitri Niks for valuable discussions on our collaborative projects and have helped me on many aspects. iv I am grateful to the U.S. National Science Foundation for providing finding for my research. I also thank American Association for the Advancement of Science for giving me the Geraldine K. Lindsay award. This award is a great encouragement to me. Last but surly not least, I would like to thank my parents and my husband, Haizhou Liu, for their unconditional love and support for me. My dissertation would never have been completed without encouragement from you all. You are the most important person to me. I love you all! Part of the text of this dissertation have been published on: “Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha).”, J. Biol. Chem. 2017; and “Electrochemical detection of dihydronicotinamide adenine dinucleotide using Al2O3-GO nanocomposite modified electrode”, Arabian Journal of Chemistry 2018. v DEDICATION To my parents and my husband vi ABSTRACT OF THE DISSERTATION Conversion of Carbon Dioxide to Formate by a Formate Dehydrogenase from Cupriavidus necator by Xuejun Yu Doctor of Philosophy, Graduate Program in Bioengineering University of California, Riverside, September 2018 Dr. Ashok Mulchandani, Co-Chairperson Dr. Xin Ge, Co-Chairperson Recently, several formate dehydrogenases have been reported to be able to catalyze the reduction of carbon dioxide to formic acid. The main challenge with these enzymes are that either the CO2-reducing activity is very low, or highly oxygen sensitive. In this dissertation, we employed an oxygen tolerant formate dehydrogenase (FdsABG) from Cupriavidus ncecator (formerly known as Ralstonia eutropha) for conversion CO2 to formic acid. We found that the enzyme is kinetically competent to catalyze the reverse -1 reaction, i.e. reduction of CO2 to formate by NADH, with a kcat of 10 s . Accumulation of formate in the reverse reaction was quantitatively account for consumption of NADH. It showed that all molybdenum- and tungsten-containing formate dehydrogenases, and probably also formylmethanofuran dehydrogenases, operate via a simple hydride transfer mechanism and were effective in catalyzing the reversible interconversion of CO2 and formate. To make the reaction of CO2 reduction more efficient and cost effective, we vii cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed in E. coli with a His-tag fused to the N terminus of fdsG subunit, and this overexpression system has simplified the FdsABG purification compared to production from native C. necator. We further combined this engineered C. necator FdsABG with glucose dehydrogenase, for continuous electron donation to the reaction of converting CO2 to formate. In dynamic electrochemistry study of FdsABG, the bioelectrocatalysis system provided electrons to FdsABG for the reduction of CO2 to formate without the expensive reducing agent, NADH. This work provided a framework for studying the electron transfer mechanisms inside of this protein and developed a highly efficient biocatalytic process for transformation of CO2 to formate that has applications as fuel for formic acid fuel-cells. viii Table of content ACKNOWLEDGEMENTS ....................................................................................... IV ABSTRACT OF THE DISSERTATION ................................................................. VII TABLE OF CONTENT ............................................................................................. IX LIST OF FIGURES ................................................................................................. XIII LIST OF TABLES....................................................................................................XIX CHAPTER 1 .................................................................................................................. 1 INTRODUCTION ......................................................................................................... 1 1.1 IMPORTANCE OF CO2 REDUCTION AND ITS METHODS ............................................... 1 1.2 BIOLOGICAL SYSTEMS ARE WIDELY APPLIED IN CARBON DIOXIDE FIXATION PROCESS 2 1.3 FORMATE DEHYDROGENASE ................................................................................... 5 1.4 CHALLENGES OF ENZYMATIC CO2 REDUCTION ......................................................... 7 1.5 SCOPE OF THIS THESIS ............................................................................................. 8 1.5.1 Chapter 2: Efficient reduction of CO2 by FdsABG from Cupriavidus necator (Ralstonia eutropha). .......................................................................................................... 9 1.5.2 Chapter 3: Synthesis of Formate from CO2 Gas Catalyzed by fdsABG and Glucose Dehydrogenase ................................................................................................................. 11 1.5.3 Chapter 4: Electrochemistry Study of FdsABG from Cupriavidus necator ................ 12 1.6 REFERENCE .......................................................................................................... 14 CHAPTER 2 ................................................................................................................ 19 ix EFFICIENT REDUCTION OF CO2 BY THE MOLYBDENUM-CONTAINING FORMATE DEHYDROGENASE FROM CUPRIAVIDUS NECATOR (RALSTONIA EUTROPHA). ....................................................................................... 19 2.1 ABSTRACT ............................................................................................................ 19 2.2 INTRODUCTION ..................................................................................................... 20 2.3 MATERIALS AND METHODS ................................................................................... 23 2.3.1 Chemicals ................................................................................................................ 23 2.3.2 Protein Preparation. .................................................................................................. 23 2.3.3 Kinetic Characterization. .......................................................................................... 24 2.3.4 Formic Acid Detection and Identification. ................................................................ 26 2.4 RESULTS ............................................................................................................... 27 2.4.1 Demonstration of CO2 as substrate for the reverse reaction. ...................................... 27 2.4.2 pH dependence of the reverse reaction. ..................................................................... 29 2.4.3 Further analysis of the reverse reaction. .................................................................... 32 2.4.4 Formic Acid Quantificaton and Identification. .......................................................... 33 2.4.5 Enzyme kinetics. ...................................................................................................... 36 2.5 DISCUSSION .......................................................................................................... 37 2.6 CONCLUSIONS....................................................................................................... 44 2.7 REFERENCES ......................................................................................................... 45 CHAPTER 3 ................................................................................................................ 51 x SYNTHESIS OF FORMATE FROM CO2 GAS CATALYZED BY AN O2- TOLERANT
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