Usage
  • 59 views
  • 31 downloads

Optimization of the reaction conditions of two enzymes for use in a carbon sequestration process, and investigation into immobilization via encapsulation within polymersomes

  • Author / Creator
    Nish, Gordon L.
  • 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 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.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R36D5PG7P
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Master's
  • Department
    • Department of Chemical and Materials Engineering
  • Specialization
    • Chemical Engineering
  • Supervisor / co-supervisor and their department(s)
    • Montemagno, Carlo (Chemical and Materials Engineering)
  • Examining committee members and their departments
    • De Klerk, Arno (Chemical and Materials Engineering)
    • Choi, Hyo-Jick (Chemical and Materials Engineering)
    • Nazemifard, Neda (Chemical and Materials Engineering)
    • Kresta, Suzanne (Chemical and Materials Engineering)