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Synthesis and Stability Evaluation of Single Enzyme Nanoparticles for Enzymatic Based Processes

Khosravi, Arezoo | 2013

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 44136 (48)
  4. University: Sharif University of Technology
  5. Department: Institute for Nanoscience and Nanotechnology
  6. Advisor(s): Vossoughi, Manoochehr; Shahrokhian, Saeed; Alemzadeh, Iran
  7. Abstract:
  8. Enzymes, the nanometric biocatalysts that play a key role in biochemical reactions of living cells and bio-organisms, offer various applications including biosensing, bioremediation and chemical synthesis. A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (Polyester-32-hydroxyl-1-carboxyl bis-MPA Dendron, Generation 5) was proposed in this project. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by TEM, DLS, GPC and FTIR spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 ºC as well as its tolerance under different elevated temperatures up to 80 ºC and also in the presence of organic solvents for 15 minutes. These significant results promise extensive applications of horseradish peroxidase (HRP) particularly in harsh environmental conditions. In the next part of project, for enzyme recovery and reusability purposes, the single enzyme nanoparticles were labelled with magnetic nanoparticles with a labeling yield of 90%. The reusability and process stability of single enzyme nanoparticles were investigated in fluorescence based sensing system for hydrogen peroxide determination as a model application. These labeled enzyme molecules showed significant process stability up to 7 recycling period in a model sensing system. Then, in the last part of project, synthesis of poly(2,5-dimethoxyanilie) (PDMA) catalyzed by HRP single enzyme nanoparticles, was studied as a function of reaction media. UV-vis, GPC and conductivity measurements were used to explain how the reaction media compositions influence the physical properties of PDMA. Nanostructured PDMA was also synthesized by oxidative polymerization in a novel and green bi-catalyst system involving HRP and ferric chloride. FTIR and UV-vis analysis confirmed the formation of emeraldine salt state of PDMA. The thermal stability of reengineered HRP granted further studies on the polymerization using bi-catalyst system at temperatures of 50 and 60 °C. The scanning electron microscopy images showed the changes in the morphology of synthesized PDMA from agglomerated nanoparticles to rod-like shapes by increasing the reaction temperature
  9. Keywords:
  10. Thermal Stability ; Horseradish Peroxidase (HPR) ; Biocatalyst ; Single Enzyme Nanoparticles ; Dendrone ; Enzyme/Polymer Hybrid Nanostructures

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