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Studying the Effective Factors and Optimizing Enzyme (α-amylase) Separation in Multi-Enzyme Systems by Agarose Column Chromatography

Ataallahi, Elahe | 2023

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 56509 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Roosta Azad, Reza; Soheila Yaghmaee; Yeganeh Sarkandi, Shahin
  7. Abstract:
  8. In all types of chromatography for the extraction and purification of proteins, a fixed substrate is needed with which the mobile phase containing the proteins interact with them. This fixed bed should contain grains that have characteristics such as sphericity, high surface area, hydrophilicity, binding power to functional groups, chemical stability, low specific absorption, reusability and low cost. Commercially, the most commonly substrates in the ranges of the molecular weight of proteins are agarose and dextran. Agar was extracted from Gracilaria salicornia seaweed and removal of ionic groups was investigated by three methods. In washing with isopropanol, the ionic groups were reduced and by removing the ionic groups, agarose with a white color, free of sulfate and a strong gel structure was obtained. The granulation of the product and the production of sepharose were done using a stirring system in the toluene, light and heavy mineral oil. Granulation in toluene system was done well, but washing with ethanol could not remove the toluene. For this reason, the passage of the aqueous phase from the grains was not done in the chromatography column. By using the light mineral oil, the solvent was washed with ethanol and removed from the environment. Physico-Chemical properties of agarose beads were investigated using FPLC device and model enzyme (alpha-amylase). The seeds were tolerated the pressure of the buffer and the enzyme was removed from the column while the activity was maintained. Model enzyme (alpha-amylase) was used in order to check the characteristics of the desired column for enzyme purification. It was done in two stages of preliminary and intermediate separation. In the preliminary stage, the acetone used in two stages. By this way, the enzyme solution be reduced to three proteins. In the stage of intermediate separation by using DEAE-sepharose column, the enzyme was purified with the order of 48.03. Then, the column prepared with agar and granulated agarose were analyzed with the model enzyme. Granulated agar can be used as a very weak cationic resin
  9. Keywords:
  10. Agarose ; Alpha-amylase Enzyme ; Gracilaria Salicornia Algae ; Sepharase ; Purification ; Agar

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