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The Use of Flavin Reductase Enzyme as Biocatalyst in Promiscuous Catalyzing Organic Reactions and Investigating Amyloid Inhibition in Proteins Using Aromatic Compounds

Masihzadeh, Zahra | 2023

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 56094 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. Enzymes are proteins that carry out chemical reactions in the form of chemo, stereo, and regioselective under mild conditions. One of the characteristics of enzymes is that they perform a specific chemical reaction on a selective substrate and under specific environmental conditions. However, in recent years, a number of enzymes have been identified to display promiscous behavior. This promiscousity includes catalytic activity, variety of substrates, and environmental conditions. Promiscous activities of enzymes can be a good starting point for creating new and more favorable engineered pathways for the production of chemical and medicinal drugs. One of the aims of this project is to exploit E. coli flavin reductase to perform promiscuous reactions. In this project, the ability of flavin reductase to perform organic reactions such as C-C and C-O bond fusions were investigated. The recombinant of flavin reductase protein was expressed and purified and its activity was measured. Then the desired reactions were monitored using TLC chromatography. The result indicated that the flavin reductase was not able to carry out the organic reactions. Proteins in order to perform their functions, they must be able to possess a three-dimensional structure appropriately and have the appropriate conformation and folding for their activities. If the proteins cannot obtain their normal structure, pr-aggregate (amyloid) could be formed. Amyloid formation has been linked to Alzheimer's disease, Parkinson's disease, and type 2 diabetes. In the second project, series of new aromatic compounds that were synthesized enzymatically including tetraketone products and monosubstituted urea compounds were investigated for their amyloid inhibitory rule. Hen Egg White Lysozyme (HEWL) was used as a model protein for the formation of amyloid fibrils. The model protein was placed in high temperature and low pH conditions for amyloid formation. A number of techniques were used to investigate the amyloid formation in the presence of organic compound; these approaches included molecular docking for identifying compounds that bind to the amyloidogenic region of lysozyme protein, gel electrophoresis for identifying accumulated intermediates, thioflavin-T for measuring the amount of amyloid formation, ANS (8-anilinonaphthalene-1-sulfonic acid) binding for measuring the amount of hydrophobic exposure, and AFM analysis for visualizing the morphology of fibril formation. Preliminary data indicated that monosubstituted urea derivatives had shown significant anti-amylogenic function
  9. Keywords:
  10. Aromatic Compounds ; Protein Misfolding ; Amyloid Formation ; Molecular Docking ; Promiscous Enzyme ; Flavin Reductase Enzyme ; Hen Egg White Lysozyme Enzyme

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