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Experimental Study of Biological Synthesis, Stabilization and Delivery of the Recombinant Protein of Chondroitinase

Askaripour, Hossein | 2020

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 52749 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Vossoughi, Manouchehr; Alemzadeh, Iran; Khajeh, Khosro
  7. Abstract:
  8. In this research, immobilization method was employed to increase the thermal stability of Chondroitinase ABC I (cABC I) enzyme. In first step, magnetite nanoparticle (Fe3O4) was selected as a support and cABC I enzyme was attached via the adsorption method. The results showed that pH=6.5, temperature 15 ˚C, enzyme-to-support mass ratio 0.75, and incubation time 4.5 hr were the appropriate conditions for immobilizing cABC I enzyme on Fe3O4 nanoparticle. It was also found that the optimum pH for free and immobilized enzymes was 7.6 and 8.0, respectively. The maximum activity of free enzyme was obtained at 25 ˚C, whereas the activity of immobilized enzyme was almost constant in the temperature range of 10-25 ˚C. In addition, the activity for immobilized cABC I was 89.1 % of that for free one. Immobilization onto magnetite nanoparticle led to an increase of the storage stability at low temperatures and a decrease of the storage stability at high temperatures. In second step, magnetite nanoparticle was coated with dextran and utilized as a support to immobilized cABC I enzyme. The results demonstrated that pH=6.3, temperature 24 ˚C, enzyme-to-support mass ratio 1.27, and incubation time 5.7 hr were the suitable conditions for immobilizing cABC I enzyme on Fe3O4-dextran nanoparticle. Also, the optimum pH for free and immobilized cABC I was acquired at pH=7.6. The activity for immobilized enzyme was 87.3 % of that for free one and immobilization on dextran-coated Fe3O4 nanoparticle enhanced the storage stability at low to high temperatures. In addition, release mechanism of cABC I enzyme from dextran-coated Fe3O4 nanoparticles obeyed the first-order kinetic model. Futhermore, XRD, SEM, and VSM analyses were used to study the characterizations of Fe3O4 and Fe3O4-dextran nanoparticles. The coating of magnetite nanoparticles with dextran as well as cABC I immobilization on Fe3O4 and Fe3O4-dextran nanoparticles were verified with FT-IR analysis
  9. Keywords:
  10. Thermal Stability ; Magnetite Nanoparticle ; Enzyme Activity ; Immobilization ; Dextrances ; Coating with Dextran ; Chondroitinase ABC I Enzyme

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