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Optimization of the Reaction Conditions of Two Enzymes for Use in a Carbon Sequestration

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Optimization of the Reaction Conditions of Two Enzymes for Use in a Carbon Sequestration Optimization of the reaction conditions of two enzymes for use in a carbon sequestration process, and investigation into immobilization via encapsulation within polymersomes by Gordon L. Nish A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department of Chemical and Materials Engineering University of Alberta © Gordon L. Nish, 2016 ii Abstract Carbon dioxide emissions from human activities contribute to an increase of greenhouse gases in the atmosphere. In nature, this gas is sequestered through the use of enzymes found in the Calvin-Benson-Bassham cycle, with useful molecules such as sugars being synthesized as products. A biomimetic approach to capturing carbon dioxide and using it to synthesize useful chemicals, by way of these enzymes in a bioprocess, has been proposed. To achieve this, active enzymes must be harvested and their kinetic properties need to be characterized. Optimal process conditions must be established, with an appropriate enzyme immobilization technique being applied to ameliorate the overall bioprocess. In this work, the phosphoribulokinase enzyme was produced in the bacterial cell platform Escherichia coli using molecular cloning techniques. The kinetic conditions affecting the rates of the enzymes ribose 5-phosphate isomerase A from E. coli, and phosphoribulokinase from Synechococcus elongatus, were locally optimized through surface response methodology and mathematical modeling. These models predicted the optimal pH levels, temperature, substrate concentration and other factors used in the assays. The rate of ribose 5-phosphate isomerase product formation under optimized conditions showed an increase of about 37 % over the measured rate under initial conditions, while that of phosphoribulokinase activity increased around 21 %. Enzyme characterization revealed Km constants for the sugar substrates to be 0.12 mM for phosphoribulokinase and 9.4 mM for ribose 5-phosphate isomerase, with half-lives of 177 minutes and 89 hours, respectively, at room temperature. Furthermore, immobilization via encapsulation within polymersomes was investigated by using a iii digestive assay and fluorescence correlation spectroscopy. The encapsulation efficiency of the enzyme Rpi was found to be about 23 %. This represents a final enzyme concentration of 1.2 µM having been encapsulated. The enzyme phosphoribulokinase was successfully purified from a bacterial platform. This enzyme and ribose 5-phosphate isomerase were characterized using reaction kinetics, resulting in calculated half-lives and Michaelis constants. Locally optimized reaction conditions were found through experimental modeling, resulting in apparent increases in the reaction rate of both of the enzymes. The enzyme ribose 5- phosphate isomerase was successfully encapsulated within polymersomes. iv Acknowledgments I would like to thank these individuals and organizations for their support in this project: Dr. Carlo Montemagno for his guidance, inspiration and tutelage Dr. Paolo Mussone for his sage advice and help in editing this thesis Dr. Hyo-Jick Choi and Dr. Arno de Klerk for help in revising this thesis Dr. Sinoj Abraham, Sumalee Salahub and Dr. Surjith Kumaran for preparing polymer, and for the help in getting the system to work Hui Qiuan (Julie) for the transmission electron microscopy work in section 2.14.3 The Climate Change and Emissions Management Corporation for their generous funding of the project My children Isaac and Mia, for behaving while their father was away at the lab My loving wife Morgan, for supporting me wholeheartedly in my endeavors v Table of Contents Chapter 1. Introduction ....................................................................................................... 1 1.1 Carbon Emissions ..................................................................................................... 1 1.2 Calvin-Benson-Bassham cycle ................................................................................. 5 1.3 Biomimicry ............................................................................................................... 8 1.4 Project overview and objectives ............................................................................... 9 Chapter 2. Literature review ............................................................................................. 11 2.1 Enzymes and kinetic characterization ..................................................................... 11 2.2 Optimization methods ............................................................................................. 25 2.3 Improving enzymatic stability ................................................................................ 29 2.4 Immobilization ........................................................................................................ 30 2.5 Encapsulation .......................................................................................................... 31 Materials and Methods ...................................................................................................... 35 2.6 Ribose 5-phosphate isomerase from E. coli ............................................................ 36 2.7 Phosphoribulokinase from Spinacia oleracea ........................................................ 41 2.8 Phosphoribulokinase from Synechoccus elongates ................................................ 50 2.9 Kinetic characterization of Rpi ............................................................................... 52 2.10 Kinetic characterization of PRK ........................................................................... 58 2.11 Optimization of enzymatic reaction conditions .................................................... 62 2.12 Polymer preparation .............................................................................................. 64 2.13 Polymersome formation and purification ............................................................. 67 2.14 Characterization of polymersomes ....................................................................... 69 2.15 Post-encapsulation experiments ............................................................................ 72 Chapter 3. Experimental results ........................................................................................ 76 3.1 Expression and purification of PRK ....................................................................... 76 vi 3.2 Kinetic characterization of Rpi and PRK................................................................ 86 3.3 Stability of enzymes ................................................................................................ 92 3.4 Local optimization of kinetic activity ..................................................................... 95 3.5 Polymer results...................................................................................................... 108 3.6 Polymersome purification and characterization.................................................... 109 3.7 Post encapsulation experimental results ............................................................... 111 Chapter 4. Discussion of results and conclusion ............................................................ 119 4.1 PRK expression and purification .......................................................................... 119 4.2 Kinetic characterization ........................................................................................ 123 4.3 Optimization results .............................................................................................. 127 4.4 Characterization of polymersomes ....................................................................... 134 4.5 Conclusion and future work .................................................................................. 140 References………………………………………………………………………………143 Appendix………………………………………………………………………………. 159 List of figures Figure 1.1 Simplified overview of enzymes involved in the regeneration of ribulose 1,5- bisphosphate in the Calvin cycle................................................................................. 7 Figure 2.1 Reaction mechanism for RpiA catalysis.......................................................... 19 Figure 2.2 Reaction mechanism of PRK........................................................................... 22 Figure 2.3 Geometry of a Box-Behnken design. .............................................................. 27 Figure 2.4 Geometry of a circumscribed central composite design.................................. 28 Figure 2.5 Schematic of enzyme encapsulation within polymersome .............................. 33 Figure 0.1 Gel images of the expression and purification of Rpi ..................................... 37 Figure 0.2 Example of raw data from Rpi kinetic assay, n=3. ......................................... 55 Figure 0.3 Example of absorbance data from a PRK kinetic assay, n=3. ......................... 59 Figure 3.1 Agarose gel of PRK gene and pET-15b vector ............................................... 76 Figure 3.2 SDS-PAGE of PRK expression in BL-21 cells. .............................................. 79 Figure 3.3 SDS-PAGE of PRK expression in C-43 cells. ................................................ 80 Figure 3.4 SDS-PAGE of PRK expression in T7 cells. .................................................... 81 vii Figure 3.5 SDS-polyacrylamide gel of ion exchange elution fractions. ..........................
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